Scalable fermentation process

ABSTRACT

This invention provides a robust fermentation process for the expression of a capsid protein of a bacteriophage which is forming a VLP by self-assembly, wherein the process is scalable to a commercial production scale and wherein the expression rate of the capsid protein is controlled to obtain improved yield of soluble capsid protein. This is achieved by combining the advantages of fed-batch culture and of lactose induced expression systems with specific process parameters providing improved repression of the promoter during the growth phase and high plasmid retention throughout the process.

FIELD OF THE INVENTION

This invention is related to the field of protein expression andfermentation technology. A process for the efficient expression ofrecombinant bacteriophage capsid protein in a bacterial host isdescribed. The process leads to high yield of recombinant capsid proteinwhich is capable of forming a virus-like particle (VLP) byself-assembly. Furthermore, the process is scalable from laboratoryscale to fermenter volumes larger than 50 litres.

BACKGROUND OF THE INVENTION

Recent vaccination strategies make use of viruses orvirus-like-particles (VLPs) to enhance the immune response towardsantigens. For example, WO02/056905 demonstrates the utility of VLPs as acarrier to present antigens linked thereto in a highly orderedrepetitive array. Such antigen arrays can cause a strong immuneresponse, in particular antibody responses, against the linked antigenand are even capable of breaking the immune system's inherent tolerancetowards self antigens. Such antigen arrays are therefore useful in theproduction of vaccines for the treatment of infectious diseases andallergies as well as for the efficient induction of self-specific immuneresponses, e.g. for the treatment of cancer, rheumatoid arthritis andvarious other diseases.

As indicated in WO02/056905 capsid proteins of bacteriophages areparticularly suited as antigen carrier. They have been shown toefficiently self-assemble into VLPs upon expression in a bacterial host(Kastelein et al. 1983, Gene 23:245-254; Kozlovskaya et al. 1986, Dokl.Akad. Nauk SSSR 287:452-455). Moreover, capsid proteins ofbacteriophages such as derived from fr (Pushko et al. 1993, ProteinEngineering 6(8)883-891), Qβ (Kozlovska et al. 1993, Gene 137:133-137;Ciliens et al. 2000, FEBS Letters 24171:1-4; Vasiljeva et al 1998, FEBSLetters 431:7-11) and MS-2 (WO92/13081; Mastico et al. 1993, Journal ofGeneral Virology 74:541-548; Heal et al. 2000, Vaccine 18:251-258) havebeen produced in bacterial hosts using inducible promoters such as thetrp promoter or a trp-T7 fusion (in the case of fr and Qb) or the tacpromoter using IPTG as inducer substance (in the case of MS-2). The useof inducible promoters is beneficial, to avoid possible toxic effects ofthe recombinant capsid protein and the metabolic burden of proteinexpression which both might reduce the growth of the bacterialexpression host and, ultimately, the yield of expressed protein.

However, the expression systems used so far for the expression of capsidproteins of bacteriophages have been applied in small scalefermentations, i.e. in laboratory scale and small batch cultures withvolumes of typically clearly below 1 litre. An scale up of these systemscomprising volumes of 50 litre and more is expected to diminish in agreat extent the respective capsid protein yield due to increasedpromoter leakage and/or lowered plasmid retention.

A further problem associated with commercially desired high-levelexpression and rapid accumulation of recombinant capsid proteins ofbacteriophages is the formation of incorrectly folded protein speciesand the formation of so called inclusion bodies, i.e. proteinaggregates, which are insoluble and which may hamper further downstreamprocesses. Thus, for bacteriophage MS-2 coat protein the formation ofprotein aggregates and of protein species which lost their ability toself-assemble to VLPs have been reported when the protein was expressedunder the control of the strong T7 promoter after IPTG induction usingthe pET expression system (Peabody & Al-Bitar 2001, Nucleic AcidResearch 29(22):e113).

High expression rates of the recombinant capsid protein may thereforehave a negative impact on the yield of correctly assembled VLPs. Theproduction of VLP-based vaccines in a commercial scale requires,therefore, the establishment of an efficient, and in particular scalablefermentation process for the expression of recombinant capsid protein ofbacteriophages leading to a product of constant quality and purityhaving the capability of self-assembling into VLPs, whereby theformation of insoluble fractions of the capsid protein is minimised oravoided.

Therefore, it is an object of the present invention to provide a processfor expression of a recombinant capsid protein of a bacteriophage whichavoids or minimizes the disadvantage or disadvantages of the prior artprocesses, and in particular, which is scalable to a commercial scaleand still leading to a product of constant quality and purity and thecapability of self-assemblance to VLPs, and wherein the formation ofinsoluble fraction of the capsid protein is minimised or avoided.

SUMMARY OF THE INVENTION

The invention relates to a process for expression of a recombinantcapsid protein of a bacteriophage, or a mutant or fragment thereof beingcapable of forming a VLP by self-assembly, said process comprising thesteps of: a) introducing an expression plasmid into a bacterial host,wherein said expression plasmid comprises an expression construct,wherein said expression construct comprises (i) a first nucleotidesequence encoding said recombinant capsid protein, or mutant or fragmentthereof, and (ii) a promoter being inducible by lactose; b.) cultivatingsaid bacterial host in a medium comprising a major carbon source;wherein said cultivating is performed in batch culture and underconditions under which said promoter is repressed by lacI, wherein saidlacI is overexpressed by said bacterial host; c.) feeding said batchculture with said major carbon source; and d.) inducing said promoterwith an inducer, wherein preferably said feeding of said batch culturewith said major carbon source is continued.

This invention provides a robust fermentation process for the expressionof a capsid protein of a bacteriophage which is forming a VLP byself-assembly, wherein the process is scalable to a commercialproduction scale and wherein the expression rate of the capsid proteinleads to improved yield of soluble capsid protein. This is, inparticular, achieved by improved repression of the promoter during thegrowth phase and high plasmid retention throughout the process. Theexpression system further avoids formation of insoluble proteinaggregates by limiting the maximum expression rate occurring during theproduction phase.

In a preferred embodiment said bacteriophage is a RNA bacteriophage.More preferably, said RNA bacteriophage is selected from the groupconsisting of: a.) bacteriophage Qβ; b.) bacteriophage AP205; c.)bacteriophage fr; d.) bacteriophage GA; e.) bacteriophage SP; f.)bacteriophage MS2; g.) bacteriophage M11; h.) bacteriophage MX1; i.)bacteriophage NL95; j.) bacteriophage f2; k.) bacteriophage PP7 and l.)bacteriophage R17. Preferably, said RNA bacteriophage is Qβ. Morepreferably said recombinant capsid protein comprises or alternativelyconsists of an amino acid sequence selected from the group consisting ofSEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, andSEQ ID NO:11. Still more preferably said recombinant capsid proteincomprises SEQ ID NO:5, most preferably said recombinant capsid proteinconsists of SEQ ID NO:5.

In a further preferred embodiment said recombinant capsid proteincomprises or alternatively consists of an amino acid sequence selectedfrom the group consisting of SEQ ID NO:12, SEQ ID NO:13, and SEQ IDNO:14. More preferably said recombinant capsid protein comprises SEQ IDNO:12, most preferably said recombinant capsid protein consists of SEQID NO:12.

In another embodiment of the present invention, said expressionconstruct comprises a first stop codon, and wherein said first stopcodon is TAA, and wherein preferably said TAA is located directly 3′ ofsaid first nucleotide sequence.

In a further embodiment said expression construct comprises a first stopcodon and a second stop codon, wherein said first stop codon is locateddirectly 3′ of said first nucleotide sequence and wherein said secondstop codon is located directly 3′ of said first stop codon, and whereinat least one of said first or second stop codon is TAA.

In a further embodiment said expression construct comprises a firstnucleotide sequence and a second nucleotide sequence, wherein said firstnucleotide sequence is encoding a recombinant capsid protein, preferablyQβ CP, or a mutant or fragment thereof, and wherein said secondnucleotide sequence is encoding any other protein, preferably the Qβ A1protein or a mutant or fragment thereof, and wherein said first and saidsecond nucleotide sequence are separated by exactly one sequence stretchcomprising at least one TAA stop codon. In a preferred embodiment saidexpression construct comprises or alternatively consists of thenucleotide sequence of SEQ ID NO:6.

In a further embodiment said expression plasmid comprises or, morepreferably, consists of the nucleotide sequence of SEQ ID NO:1.

In one embodiment of the invention said promoter is selected from thegroup consisting of the a.) tac promoter; b.) trc promoter; c.) ticpromoter; d.) lac promoter; e.) lacUV5 promoter; f.) P_(syn) promoter;g.) lpp^(a) promoter; h.) lpp-lac romoter; i.) T7-lac promoter; j.)T3-lac promoter; k.) T5-lac promoter; and l.) a promoter having at least50% sequence homology to SEQ ID NO:2. In a preferred embodiment saidpromoter has at least 50%, 60%, 70%, 80, 90, or 95%, preferably 98 to100%, most preferably 99% sequence homology to SEQ ID NO:2. In a furtherpreferred embodiment said promoter is selected from the group consistingof tic promoter, trc promoter and tac promoter. Even more preferablysaid promoter is the tac promoter. Most preferably said promotercomprises or alternatively consists of the nucleotide sequence of SEQ IDNO:2.

In one embodiment said major carbon source is glucose or glycerol,preferably glycerol.

In one embodiment said feeding of said batch culture is performed with aflow rate, wherein said flow rate increases with an exponentialcoefficient μ, and wherein preferably said exponential coefficient μ isbelow μ_(max).

In a further embodiment said inducing of said promoter is performed byco-feeding said batch culture with said inducer, preferably lactose andsaid major carbon source, preferably glycerol, at a constant flow rate.

In a further embodiment said inducing of said promoter is performed byco-feeding said batch culture with said inducer, preferably lactose andsaid major carbon source, preferably glycerol, at an increasing flowrate.

In a further embodiment said inducer is lactose, wherein preferably saidlactose and said major carbon source are co-fed to said batch culture ina ratio of about 2:1 to 1:4 (w/w).

In a further embodiment said inducer is IPTG wherein preferably theconcentration of said IPTG said medium is 0.001 to 5 mM, preferably0.001 to 1 mM, more preferably 0.005 to 1 mM, still more preferably0.005 to 0.5 mM. In a very preferred embodiment said concentration ofIPTG is about 0.01 mM, most preferably 0.01 mM.

In one embodiment said lacI is overexpressed by said bacterial host,wherein said overexpression is caused by lacI^(q) or lacQ1, preferablyby lacI^(q). In one embodiment said bacterial host comprises saidlacI^(q) gene or said lacQ1 gene, preferably said lacI^(q) gene on itschromosome. In a further preferred embodiment said bacterial hostcomprises said lacI^(q) gene or said lacQ1 gene, preferably saidlacI^(q) gene on a plasmid, preferably on a high copy number plasmid. Ina further preferred embodiment said bacterial host comprises saidlacI^(q) gene or said lacQ1 gene, preferably said lacI^(q) gene on saidexpression plasmid.

In one embodiment said bacterial host is selected from the groupconsisting of the strains E. coli RB791, E. coli DH20 and E. coli Y1088.Preferably said bacterial host is E. coli RB791.

In one embodiment said bacterial host comprises β-galactosidaseactivity.

In one embodiment said cultivating and said feeding of said batchculture and said inducing of said promoter is performed at a temperaturewhich is below the optimal growth temperature of said bacterial host.Preferably said temperature is between 23° C. and 35° C., morepreferably between 25 and 33° C., even more preferably between 27 and32° C., still more preferably between 28 and 31° C. Even more preferablysaid temperature is about 30° C., most preferably said temperature is30° C.

In one embodiment said cultivating and said feeding of said batchculture is performed at a temperature which is below the optimal growthtemperature of said bacterial host, wherein preferably said temperatureis between 23° C. and 35° C., more preferably between 25 and 33° C.,even more preferably between 27 and 32° C., still more preferablybetween 28 and 31° C., even more preferably said temperature is about30° C., most preferably said temperature is 30° C., and said inducing ofsaid promoter is performed at the optimal growth temperature of thebacterial host, preferably at about 37° C.

In one embodiment said cultivating and said feeding of said batchculture and said inducing of said promoter is performed in the absenceof an antibiotic.

In a specific embodiment said expression plasmid comprises oralternatively consists of the nucleotide sequence of SEQ ID NO:1, saidmajor carbon source is glycerol, said feeding of said batch culture isperformed with a flow rate, wherein said flow rate increases with anexponential coefficient μ, and wherein said exponential coefficient μ isbelow μ_(max), said inducing of said promoter by co-feeding said batchculture is performed with a constant flow rate, wherein lactose andglycerol are co-fed to the batch culture in a ratio of about 2:1 toabout 1:4 (w/w), preferably about 1:1 to about 1:4 (w/w), mostpreferably about 1:3 (w/w), and wherein said cultivating and feeding ofsaid batch culture and said inducing of said promoter is performed at atemperature between 27 and 32° C., preferably about 30° C., mostpreferably 30° C.

In a further specific embodiment said expression plasmid comprises oralternatively consists of the nucleotide sequence of SEQ ID NO:30, saidmajor carbon source is glycerol, said feeding of said batch culture isperformed with a flow rate, wherein said flow rate increases with anexponential coefficient μ, and wherein said exponential coefficient μ isbelow μ_(max), said inducing of said promoter by co-feeding said batchculture is performed with a constant flow rate, wherein lactose and saidmajor carbon source are co-fed to the batch culture in a ratio of about2:1 to about 1:4 (w/w), preferably about 1:1 to about 1:4 (w/w), mostpreferably about 1:3 (w/w), and wherein said cultivating and feeding ofsaid batch culture and said inducing of said promoter is performed at atemperature between 27 and 32° C., preferably about 30° C., mostpreferably 30° C.

DESCRIPTION OF THE FIGURE

FIG. 1: Fermentation profile with pTac-nSD-Qb-mut (SEQ ID NO:1) in RB791in 21 culture. Co-feeding during production phase was performed withmedium containing 20% glycerol and 20% lactose. Shown are glycerolconcentration [g/l] (circles); lactose concentration [g/l] (triangles);β-Gal activity [U/ml*OD=1] (squares) and OD600 (diamonds) plottedagainst the process time [h].

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs.

“about”: within the meaning of the present application the expressionabout shall have the meaning of +/−10%. For example about 100 shall mean90 to 110.

“promoter which is inducible by lactose” as used herein refers to apromoter which comprises regulatory elements of the lac operon. Suchpromoters are repressed by lacI and can be induced by lactose or thesynthetic inducer IPTG. The skilled person is aware that induction of apromoter by lactose requires β-galactosidase activity in the bacterialhost.

“located directly 3′”: a nucleotide sequence N2 which is locateddirectly 3′ of another nucleotide sequence N1 refers to a continuoussequence having the conformation 5′-N1-N2-3′ wherein N1 and N2 aredirectly connected and not separated by additional sequence elements.

“sequence stretch”: as used herein the term “sequence stretch” refers toa continuous nucleotide sequence which consists of less than 50,preferably less than 20, more preferably less than 10, even morepreferably less than 5 nucleotides. In a further preferred embodimentthe sequence stretch comprises or alternatively consists of at leastone, preferably one, TAA stop codon. In another embodiment the sequencestretch comprises or alternatively consists of at least one, preferablyone, TAA and at least one, preferably one, TGA stop codon. In furtherpreferred embodiment the sequence stretch comprises or alternativelyconsists of SEQ ID NO:32.

“bacterial host”: as used herein the term “bacterial host” refers to abacterial organism which is hosting or capable of hosting an expressionplasmid of the invention, wherein “hosting” involves the replication ofthe expression plasmid and maintenance of the expression plasmid duringcell division.

“culture”: in the context of the instant invention a “culture” comprisesa bacterial host in a medium (“bacterial culture”), wherein typicallysaid medium is supporting the growth of said bacterial host.

“batch culture” as used herein relates to a culture, i.e. a bacterialhost in a medium, wherein said culture constitutes a closed system, i.e.typically and preferably no addition or removal of medium takes placeduring the cultivation time. Therefore, in contrast to a continuousculture, typically and preferably the density of the bacterial host inthe batch culture continuously increases with progressing cultivationtime. Batch culture does not exclude the addition of compounds requiredfor the control of the process, such as, for example, inducer, oxygen,and alkali or acid to control the pH.

“fed batch culture”: as used herein is a culture which is supplied withadditional medium comprising a substrate, preferably the major carbonsource of the bacterial host (feed or co-feed medium). In the context ofthe application this process is referred to by the terms “feeding saidbatch culture” (medium comprises the major carbon source) and“co-feeding said batch culture” (medium comprises the major carbonsource and the inducer, preferably lactose). Typically and preferably,no removal of medium except for analytical purposes takes place duringcultivation time of a fed batch culture.

“Preculture”: a culture, preferably a batch culture, which is used toproduce the inoculum for a culture of a larger volume, e.g. the culturein which the recombinant capsid protein is produced (productionculture). A preculture can be performed in two or more steps, wherein asecond preculture is inoculated with a first preculture etc. to producea sufficiently large inoculum for the production culture. The firstand/or subsequent precultures may comprise an antibiotic to improveplasmid stability.

“substrate”: as used herein refers to a compound in the culture mediumwhich contributes to the carbon and energy supply of the bacterial host.The terms “substrate” therefore encompasses any compound contained inthe medium contributing to the carbon supply of the bacterial host.Typical substrates for bacteria are sugar, starch, glycerol, acetate andany other organic compound which can be metabolized by bacteria.Therefore, the term “substrate” includes the major carbon source butalso, for example, lactose.

“Major carbon source” as used herein refers to the compound in theculture medium which contributes most to the carbon and energy supply ofthe bacterial host during the growth phase. The major carbon source thusis the major substrate of the bacterial host. The major carbon source istypically a sugar such as sucrose or glucose, or glycerol, andpreferably glucose or glycerol. Though lactose could in principal act asa major carbon source for a bacterial host, in the context of theinstant invention the term “major carbon source” typically andpreferably does not include lactose.

Phases of the process of the invention: The process of the invention ischaracterised by different phases which refer to different physiologicalconditions of the bacterial host with respect to its growth and therepression/induction status of the expression construct.

“Growth phase”: The growth phase is initiated by said cultivating saidbacterial host in a medium. The growth phase is preferably characterizedby conditions under which the promoter driving the expression of therecombinant capsid protein is repressed and the growth phase isterminated with said inducing said promoter with an inducer. The growthphase can be further divided in a “batch phase” and a “feed phase”. Saidbatch phase is initiated by said cultivating said bacterial host in amedium. The batch phase comprised a “lag phase” during which thebacterial host is not yet growing or growing with a non-exponentialrate, typically and preferably a linear rate. The growth phase furthercomprises an “exponential growth phase” which directly follows the lagphase. No feeding of said culture takes place during the batch phase,thus the exponential growth phase is terminated by the consumption ofthe substrate by the bacterial host. The growth phase further comprisesa “feed phase” which is directly following the batch phase and which isinitiated by said feeding of said batch culture with said major carbonsource. The feed phase is characterised by a growth rate of thebacterial host which is directly dependent on the flow rate of the feedmedium containing the major carbon source.

“production phase”: The growth phase is followed by the production phasewhich is initiated by said inducing said promoter with an inducer,wherein typically and preferably said feeding of said batch culture withsaid major carbon source is continued.

“Conditions under which the promoter is repressed”: it is to beunderstood that the repression of a promoter is an equilibrium offormation and dissociation of the repressor-operator complex and thateven stringently repressed promoters may show a certain expression ratealso in the absence of their inducer. Therefore, as used within theapplication the term “conditions under which the promoter is repressed”relates to conditions, wherein at the end of the growth phase, i.e.directly before the addition of inducer to the culture, the recombinantcapsid protein is expressed to a level which does not exceed aconcentration in the medium of 200 mg/l, preferably 150 mg/l, morepreferably 100 mg/l, as determined by the HLPC method of Example 17.Most preferably, the concentration of the recombinant protein is belowthe detection level of said method.

“Inducer”: within the meaning of the in invention the term “inducer”relates to any substance which directly or indirectly interacts with aninducible promoter and thereby facilitates expression from saidpromoter; for example, inducers of “a promoter inducible by lactose”,such as the lac or tac promoter, are IPTG, lactose and allolactose.

“Coat protein”/“capsid protein”: The term “coat protein” and theinterchangeably used term “capsid protein” within this application,refers to a viral protein, preferably a subunit of a natural capsid of avirus, preferably of a RNA bacteriophage, which is capable of beingincorporated into a virus capsid or a VLP. For example, the specificgene product of the coat protein gene of RNA bacteriophage Qβ isreferred to as “Qβ CP”, whereas the “coat proteins” or “capsid proteins”of bacteriophage Qβ comprise the “Qβ CP” as well as the A1 protein.

“Recombinant capsid protein”: A capsid protein which is synthesised by arecombinant host cell.

“Polypeptide”: As used herein the term “polypeptide” refers to a polymercomposed of amino acid residues, generally natural amino acid residues,linked together through peptide bonds. Although a polypeptide may notnecessarily be limited in size, the term polypeptide is often used inconjunction with peptide of a size of about ten to about 50 amino acids.

“Protein”: As used herein, the term protein refers to a polypeptidegenerally of a size of above 20, more particularly of above 50 aminoacid residues. Proteins generally have a defined three dimensionalstructure although they do not necessarily need to, and are oftenreferred to as folded, in opposition to peptides and polypeptides whichoften do not possess a defined three-dimensional structure, but rathercan adopt a large number of different conformations, and are referred toas unfolded.

“Recombinant host cell”: As used herein, the term “recombinant hostcell” refers to a host cell into which one or more nucleic acidmolecules of the invention have been introduced.

“Recombinant VLP”: The term “recombinant VLP”, as used herein, refers toa VLP that is obtained by a process which comprises at least one step ofrecombinant DNA technology. The term “VLP recombinantly produced”, asused herein, refers to a VLP that is obtained by a process whichcomprises at least one step of recombinant DNA technology. Thus, theterms “recombinant VLP” and “VLP recombinantly produced” areinterchangeably used herein and should have the identical meaning.

“RNA-bacteriophage”: As used herein, the term “RNA-bacteriophage” refersto RNA viruses infecting bacteria, preferably to single-strandedpositive-sense RNA viruses infecting bacteria.

“Virus-like particle (VLP)”: as used herein, the term “virus-likeparticle” refers to a structure resembling a virus particle or it refersto a non-replicative or non-infectious, preferably a non-replicative andnon-infectious virus particle, or it refers to a non-replicative ornon-infectious, preferably a non-replicative and non-infectiousstructure resembling a virus particle, preferably a capsid of a virus.The term “non-replicative”, as used herein, refers to being incapable ofreplicating the genome comprised by the VLP. The term “non-infectious”,as used herein, refers to being incapable of entering the host cell.Preferably a virus-like particle in accordance with the invention isnon-replicative and/or non-infectious since it lacks all or part of theviral genome or genome function. Typically a virus-like particle lacksall or part of the replicative and infectious components of the viralgenome. A virus-like particle in accordance with the invention maycontain nucleic acid distinct from their genome. A typical and preferredembodiment of a virus-like particle in accordance with the presentinvention is a viral capsid such as the viral capsid of thecorresponding virus, bacteriophage, preferably RNA-phage. The terms“viral capsid” or “capsid”, refer to a macromolecular assembly composedof viral protein subunits. Typically, there are 60, 120, 180, 240, 300,360 and more than 360 viral protein subunits. Typically and preferably,the interactions of these subunits lead to the formation of viral capsidor viral-capsid like structure with an inherent repetitive organization,wherein said structure is, typically, spherical or tubular. For example,the capsids of RNA bacteriophages or HBcAgs have a spherical form oficosahedral symmetry.

“Virus-like particle of a RNA bacteriophage”: As used herein, the term“virus-like particle of a RNA bacteriophage” refers to a virus-likeparticle comprising, or preferably consisting essentially of orconsisting of coat proteins, mutants or fragments thereof, of a RNAbacteriophage. In addition, virus-like particle of a RNA bacteriophageresembling the structure of a RNA bacteriophage, being non replicativeand/or non-infectious, and lacking at least the gene or genes encodingfor the replication machinery of the RNA bacteriophage, and typicallyalso lacking the gene or genes encoding the protein or proteinsresponsible for viral attachment to or entry into the host. PreferredVLPs derived from RNA bacteriophages exhibit icosahedral symmetry andconsist of 180 subunits. A preferred method to render a virus-likeparticle of a RNA bacteriophage non replicative and/or non-infectious isby genetic manipulation.

one, a, or an: When the terms “one,” “a,” or “an” are used in thisdisclosure, they mean “at least one” or “one or more,” unless otherwiseindicated.

“Sequence identity”: The amino acid sequence identity of polypeptidescan be determined conventionally using known computer programs such asthe Bestfit program. When using Bestfit or any other sequence alignmentprogram, preferably using Bestfit, to determine whether a particularsequence is, for instance, 95% identical to a reference amino acidsequence, the parameters are set such that the percentage of identity iscalculated over the full length of the reference amino acid sequence andthat gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed. This aforementionedmethod in determining the percentage of identity between polypeptides isapplicable to all proteins, polypeptides or a fragment thereof disclosedin this invention.

“Sequence homology”: The homology of nucleotide sequences can forexample be determined by the program blastn which is an implementationof the BLAST algorithm, preferably using the default settings of thesoftware.

“Fragment of a protein”, in particular fragment of a recombinant proteinor recombinant coat protein, as used herein, is defined as apolypeptide, which is of at least 70%, preferably at least 80%, morepreferably at least 90%, even more preferably at least 95% the length ofthe wild-type recombinant protein, or coat protein, respectively andwhich preferably retains the capability of forming VLP. Preferably thefragment is obtained by at least one internal deletion, at least onetruncation or at least one combination thereof. Further preferably thefragment is obtained by at most 10, at most 9, at most 8, at most 7, atmost 6, at most 5, at most 4, at most 3 or at most 2 internal deletions;by at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, atmost 4, at most 3 or at most 2 truncations; or by at most 3, preferablyat most 2, most preferably by exactly one combination thereof. Mostpreferably the fragment is obtained by exactly one internal deletion,exactly one truncation or by a combination thereof.

The term “fragment of a recombinant protein” or “fragment of a coatprotein” shall further encompass polypeptide, which has at least 80%,preferably 90%, even more preferably 95% amino acid sequence identitywith the “fragment of a recombinant protein” or “fragment of a coatprotein”, respectively, as defined above and which is preferably capableof assembling into a virus-like particle.

The term “mutant recombinant protein” or the term “mutant of arecombinant protein” as interchangeably used in this invention, or theterm “mutant coat protein” or the term “mutant of a coat protein”, asinterchangeably used in this invention, refers to a polypeptide havingan amino acid sequence derived from the wild type recombinant protein,or coat protein, respectively, wherein the amino acid sequence is atleast 80%, preferably at least 85%, 90%, 95%, 97%, or 99% identical tothe wild type sequence and preferably retains the ability to assembleinto a VLP.

The invention is related to an efficient fermentation process for theproduction of a VLP of a bacteriophage. The process is improved withrespect to yield of the VLP and can be scaled up to a commercialproduction scale. The process encompasses the expression of recombinantcapsid protein of bacteriophages in a bacterial host under conditionswhich allow the capsid protein to self-assemble into VLPs spontaneously.

Specific examples of VLPs which can be produced by the process of theinvention are VLPs of bacteriophages, preferably RNA bacteriophages. Inone preferred embodiment of the invention, the virus-like particle ofthe invention comprises, consists essentially of, or alternativelyconsists of, recombinant coat proteins, mutants or fragments thereof, ofa RNA-phage. Preferably, the RNA-phage is selected from the groupconsisting of a) bacteriophage Qβ; b) bacteriophage R17; c)bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f)bacteriophage MS2; g) bacteriophage M11; h) bacteriophage MX1; i)bacteriophage NL95; k) bacteriophage f1; l) bacteriophage PP7 and m)bacteriophage AP205.

In one preferred embodiment of the invention, VLPs are producedcomprising coat protein, mutants or fragments thereof, of RNAbacteriophages, wherein the coat protein has an amino acid sequenceselected from the group consisting of: (a) SEQ ID NO:5 referring to QβCP; (b) a mixture of SEQ ID NO:5 and SEQ ID NO:15 (Qβ A1 protein); (c)SEQ ID NO:16 (R17 capsid protein); (d) SEQ ID NO:17 (fr capsid protein);(e) SEQ ID NO:18 (GA capsid protein); (f) SEQ ID NO:19 (SP capsidprotein); (g) a mixture of SEQ ID NO:19 and SEQ ID NO:20; (h) SEQ IDNO:21 (MS2 capsid protein); (i) SEQ ID NO:22 (M11 capsid protein); (j)SEQ ID NO:23 (MX1 capsid protein); (k) SEQ ID NO:24 (NL95 capsidprotein); (l) SEQ ID NO:25 (f2 capsid protein); (m) SEQ ID NO:26 (PP7capsid protein); and (n) SEQ ID NO:12 (AP205 capsid protein).

Upon expression in E. coli, the N-terminal methionine of Qβ coat proteinis usually removed (Stoll, E. et al., J. Biol. Chem. 252:990-993(1977)). VLP composed of Qβ coat proteins where the N-terminalmethionine has not been removed, or VLPs comprising a mixture of Qβ coatproteins where the N-terminal methionine is either cleaved or presentare also within the scope of the present invention.

In one preferred embodiment of the invention, the VLP is a mosaic VLPcomprising or alternatively consisting of more than one amino acidsequence, preferably two amino acid sequences, of coat proteins, mutantsor fragments thereof, of a RNA bacteriophage.

In one very preferred embodiment, the VLP comprises or alternativelyconsists of two different coat proteins of a RNA bacteriophage, said twocoat proteins have an amino acid sequence of SEQ ID NO: 5 and SEQ IDNO:15, or of SEQ ID NO:19 and SEQ ID NO:20.

In preferred embodiments of the present invention, the produced VLPcomprises, or alternatively consists essentially of, or alternativelyconsists of recombinant coat proteins, mutants or fragments thereof, ofthe RNA-bacteriophage Qβ, fr, AP205 or GA.

In one preferred embodiment, the VLP is a VLP of RNA-phage Qβ. Thecapsid or virus-like particle of Qβ shows an icosahedral phage-likecapsid structure with a diameter of 25 nm and T=3 quasi symmetry. Thecapsid contains 180 copies of the coat protein, which are linked incovalent pentamers and hexamers by disulfide bridges (Golmohammadi, R.et al., Structure 4:543-5554 (1996)).

Preferred virus-like particles of RNA bacteriophages, in particular ofQβ and fr in accordance of this invention are disclosed in WO 02/056905,the disclosure of which is herewith incorporated by reference in itsentirety. Particular Example 18 of WO 02/056905 gave detaileddescription of preparation of VLP particles from Qβ.

In another preferred embodiment, the VLP is a VLP of RNA bacteriophageAP205. Assembly-competent mutant forms of AP205 VLPs, including AP205coat protein with the substitution of proline at amino acid 5 tothreonine, may also be used in the practice of the invention and leadsto other preferred embodiments of the invention. WO 2004/007538describes, in particular in Example 1 and Example 2, how to obtain VLPcomprising AP205 coat proteins, and hereby in particular the expressionand the purification thereto. WO 2004/007538 is incorporated herein byway of reference.

In one preferred embodiment, the VLP comprises or consists of a mutantcoat protein of a virus, preferably a RNA bacteriophage, wherein themutant coat protein has been modified by removal of at least one lysineresidue by way of substitution and/or by way of deletion. In anotherpreferred embodiment, the VLP of the invention comprises or consists ofa mutant coat protein of a virus, preferably a RNA bacteriophage,wherein the mutant coat protein has been modified by addition of atleast one lysine residue by way of substitution and/or by way ofinsertion. The deletion, substitution or addition of at least one lysineresidue allows varying the degree of coupling with an antigen.

VLPs or capsids of Qβ coat protein display a defined number of lysineresidues on their surface, with a defined topology with three lysineresidues pointing towards the interior of the capsid and interactingwith the RNA, and four other lysine residues exposed to the exterior ofthe capsid.

Qβ mutants, of which exposed lysine residues are replaced by argininesare also encompassed by the present invention. Preferably these mutantcoat proteins comprise or alternatively consist of an amino acidsequence selected from the group of a) Qβ-240 (SEQ ID NO:7, Lys13→Arg);b) Qβ-243 (SEQ ID NO:8, Asn10→Lys); c) Qβ-250 (SEQ ID NO:9, Lys2→Arg);d) Qβ-251 (SEQ ID NO:10, Lys16→Arg); and e) Qβ-259 (SEQ ID NO:11,Lys2→Arg, Lys16→Arg). The construction, expression and purification ofthe above indicated Qβ mutant coat proteins, mutant Qβ coat protein VLPsand capsids, respectively, are described in WO02/056905. In particularis hereby referred to Example 18 of above mentioned application.

In a further preferred embodiment the recombinant capsid protein is acapsid protein of bacteriophage AP205 having the amino acid sequencedepicted in SEQ ID NO:12 or a mutation thereof, which is capable offorming a VLP, for example the proteins AP205P5T (SEQ ID NO:13) or AP205N14D (SEQ ID NO:14.).

In a very preferred embodiment said recombinant capsid protein iscomposed of the 133 amino acid coat protein C of E. coli RNAbacteriophage Qβ comprising or preferably consisting of the amino acidsequence depicted in SEQ ID NO:5, wherein preferably said recombinantcapsid protein is capable of forming a VLP by self-assembly.

In one embodiment, the expression construct comprises a first stop codonand a second stop codon, wherein said first stop codon is locateddirectly 3′ of said first nucleotide sequence and wherein said secondstop codon is located directly 3′ of said first stop codon, and whereinat least one of said first or second stop codon is TAA. For example,plasmid pTac-nSDAP205 (SEQ ID NO:30) comprises the naturally occurringTAA stop codon as a first stop codon and an additional TGA stop codondirectly 3′ of the first stop codon.

In a preferred embodiment the expression construct comprises a firstnucleotide sequence and a second nucleotide sequence, wherein said firstnucleotide sequence is encoding a recombinant capsid protein, preferablyQβ CP, or a mutant or fragment thereof, most preferably SEQ ID NO:5, andwherein said second nucleotide sequence is encoding any other protein,preferably the Qβ A1 protein or a mutant or fragment thereof, mostpreferably SEQ ID NO:15, and wherein said first and said secondnucleotide sequence are separated by exactly one sequence stretchcomprising at least one TAA stop codon. In one embodiment said TAA stopcodon is generated by replacing the naturally occurring stop codon,preferably TGA by the sequence TAA. Alternatively and more preferrablysaid TAA stop codon is generated by replacing the naturally occurringstop codon, preferably TGA by the sequence TAATGA (SEQ ID NO:32).

For example, the region of Qβ gene C corresponds to the NCBI GenBankAcc. No. M99039 (nucleotides 46-1062). Gene C contains a firstnucleotide sequence encoding the 133-amino acid Qβ coat protein (SEQ IDNO:5) and a second nucleotide sequence encoding the 329-amino acid readthrough protein A1 (SEQ ID NO:15). Nucleotides 1-399 of SEQ ID NO:6(nucleotides 46-444 of NCBI GenBank Ace. No. M99039) correspond to saidfirst nucleotide sequence encoding the 133-amino acid Qβ CP, Nucleotides400 to 402 of SEQ ID NO:6 correspond to the strong TAA stop codon andnucleotides 403 to 405 of SEQ ID NO:6 to the leaky TGA stop codon, whichis followed by said second nucleotide sequence (Qβ A1). Surprisingly, itwas found that the presence of the nucleotide sequence relating to A1 inthe expression construct results in higher RNA stability and, thus, inimproved yield of Qβ CP and VLP as compared to a construct wherein theA1 sequence is deleted.

The expression of a recombinant protein can significantly reduce thegrowth rate of the bacterial host due to toxic effects of theaccumulating protein and due to the metabolic burden caused by theprotein synthesis. In particular cell lysis and low plasmid retentionmay occur. Inducible promoters provide for the possibility to separatethe growth phase from the production phase of a fermentation process.Inducible promoters are repressed by a repressor molecule during thegrowth phase of the bacterial host and are induced by exposing thebacterial host to inductive conditions during the production phase.Inducible promoters therefore allow the bacterial host to grow fast,preferably exponentially during the growth phase and to reach high celldensities. Thus, inducible promoters provide for high yield of theexpression product at the end of the production phase. Therefore, theusage of inducible promoters for the expression of recombinant proteinis preferred.

A well known example for an inducible promoter is the lac promoter whichforms part of the lac operon and which can be induced by addition oflactose or the strong synthetic inducer isopropylthio-β-D-galactosid(IPTG) to the growth medium of the bacterial host. Donavan et al. 2000(Can. J. Microbiol. 46:532-541) report on an improved process for theexpression of a monoclonal antibody fragment under the control of thelac promoter. Further examples of inducible promoters are provided intable 1 of Makrides 1996 (Microbiological Reviews, p. 512-538).

A typical drawback of expression systems based on inducible promoters isthe “leakiness” of the promoter, meaning that the promoter is onlyinsufficiently repressed and causes a certain expression rate of therecombinant protein during the growth phase. This typically leads to areduced cell density or to plasmid instability and, as a consequence, toreduced yield of the recombinant protein Makrides 1996 (MicrobiologicalReviews, p. 512-538). An example of a promoter which is prone toinsufficient repression is the VHb promoter which is repressed underhigh oxygen conditions and induced upon oxygen depletion.

For the purpose of the invention promoters are preferred which arestringently repressed. In one embodiment the promoter is repressed bythe repressor lacI. Examples of such promoters are disclosed in Makrides1996 (Microbiol. Rev. 60:512-538), Goldstein & Doi 1995 (BiotechnologyAnnual Review 1:105-128), Hannig & Makrides 1998 (TIBTECH 16:54-60) andStevens 2000 (Structures 8, R177-R185). In a preferred embodiment thepromoter is inducible by lactose, more preferably it is selected fromthe group consisting of lac, lacUV5, tac, trc, P_(syn) lpp^(a), lpp-lac,T7-lac, T3-lac, and T5-lac. Especially preferred for the purpose of theinvention is the tac promoter (SEQ ID NO:2) or a mutation or variantthereof. Within the scope of the invention are mutants or truncated ordeleted variants of the promoter having a sequence homology with SEQ IDNO:2 which is at least 50%, 60%, 70%, 80, 90, or 95%, preferably 98 to100%, most preferably 99%. Wherein the promoter strength of such mutatedtruncated or deleted variant is comparable to that of the promoter ofSEQ ID NO:2. The skilled person will be able to determine the promoterstrength of a given sequence by comparative expression studies usingstandard methods. In a specific embodiment of the invention the promoterdriving the expression of the recombinant capsid protein comprises oralternatively consists of SEQ ID NO:2. The tac promoter is a fusionproduct of the −10 region of the lacUV5 promoter and the −35 region ofthe trp promoter and combines the high transcription efficiency of trpwith the regulatory elements of the lac promoter (de Boer et al. 1983,PNAS 80:21-25; Amann et al. 1983 Gene 25:167-178). It provides forsufficiently high expression rates and high protein yield while avoidingthe formation of insoluble or incorrectly folded recombinant proteinwhich may occur with stronger promoters, such as the T7 promoter. Thetrc and the tic promoter are mutated versions of the promoter (Brosiuset al. 1985, The Journal of Biological Chemistry 260(6):3539-3541). In afurther preferred embodiment the promoter is selected from the groupconsisting of tic, trc and tac.

For the construction of an expression construct for the purpose of theinvention the promoter is operably linked to said first nucleotidesequence encoding the recombinant capsid protein via a ribosome bindingsite (Shine-Dalgarno sequence, SD), typically comprising an ATG startcodon at its 3′ end. Suitable Shine-Dalgarno sequences for the purposeof the invention are well known in the art (Dalbøge et al. 1988, DNA7(6):399-405) Ringquist et al. 1992, (Mol. Micr. 6:1219-1229). In oneembodiment of the invention the expression construct comprises the SDsequence of Dalbøge et al. 1988 (DNA 7(6):399-405) which is depicted inSEQ ID NO:4. In another, preferred, embodiment the expression constructcomprises a Shine-Dalgarno sequence of Ringquist et al. 1992 (Mol. Micr.6:1219-1229, SEQ ID NO:3, nSD). Surprisingly, it was found that SEQ IDNO:3 is particularly suited for the purpose of the invention because itresults in improved expression levels and improved yield of recombinantcapsid protein. SEQ ID NO:3 is especially suited to enhance theexpression of AP205 capsid protein. In a preferred embodiment of theinvention the expression construct comprises a Shine-Dalgarno sequenceselected form the group consisting of SEQ ID NO:3 and SEQ ID NO:4,preferably said Shine-Dalgarno sequence is SEQ ID NO:3.

Transcriptional terminators are functional elements of expressionconstructs. The skilled person will be able to choose a suitableterminator sequence form a wide range of sources. In a preferredembodiment of the invention said expression construct comprises aterminator sequence, wherein preferably said terminator sequence isoperably linked to said first nucleotide sequence, wherein furtherpreferably said terminator sequence is the rRNB terminator sequence,most preferably SEQ ID NO:28.

For the purpose of plasmid selection the skilled person will typicallyuse an antibiotic resistance marker gene. Examples of antibioticresistance genes which are widely used in the art and which are suitablefor the purpose of the invention are resistance genes against theantibiotics ampicillin, tetracyclin and kanamycin. The use of kanamycinas a selective agent in the frame of a process for the production of aVLP is generally preferred because of the lower allergenic potential ofkanamycin as compared to alternative antibiotics and because of thelower safety concerns resulting thereof for the use of the VLP as avaccine. Furthermore, kanamycin provides better plasmid retention ascompared to alternative antibiotics such as ampicillin. The kanamycin3′-phosphotransferase gene (SEQ ID NO:29) which is derived from thetransposon Tn903 is therefore a particularly useful selectable markergene.

The addition of antibiotics to the medium is generally undesirable in acommercial production process for cost and safety reasons. In thecontext of the invention antibiotics, preferably kanamycin, aretypically and preferably used for the selection of the expressionstrain. Media used in the production process are essentially free ofantibiotics, in particular kanamycin. However, addition of an antibioticto precultures used to produce the inoculum for the production culturecan improve plasmid retention throughout the process (Example 10).

The skilled person will create expression plasmids comprising expressionconstructs which are useful for the production of VLPs of bacteriophagesby combining the genetic elements described above applying standardmethods of molecular biology. Particularly useful expression plasmidsfor the purpose of the invention are pTac-nSDQb-mut (SEQ ID NO:1) forthe production of Qβ VLP and pTac-nSDAP205 (SEQ ID NO:30) for theproduction of AP205 VLP. The construction of these specific expressionplasmids is described in detail in the Examples section.

The expression plasmids are transformed to a bacterial expression hostby any method known in the art, preferably by electroporation.Individual clones of the host comprising the expression plasmid areselected for maximal expression of the recombinant capsid protein bySDS-PAGE after cell lysis. Selected clones of the expression hostcomprising the expression plasmid can be stored as frozen glycerolcultures.

Said bacterial host can be chosen from any bacterial strain capable ofreplicating and maintaining said expression plasmid during celldivision. Preferred bacterial hosts are Escherichia coli strains havingthe specific features described in the following sections.

The repression of the promoter is improved by overexpression of therepressor by the bacterial host. In one embodiment said cultivating ofsaid bacterial host is performed in batch culture and under conditionsunder which said promoter is repressed by lacI. In a preferredembodiment the gene causing overexpression of said lacI in saidbacterial host is located on a plasmid, preferably on said expressionplasmid. Alternatively, said gene is located on a seperate plasmidcontained in said bacterial host, wherein said seperate plasmidpreferably is a high copy number plasmid. Alternatively, and mostpreferably said gene is located on the chromosome of said bacterialhost.

One example of a gene causing overexpression of lacI is lacI^(q)(Menzella et al. 2003, Biotechnology and Bioengineering 82(7)809-817)which is a single CG to TA change at −35 of the promoter region of lacIwhich causes a 10 fold increase in LacI expression. A further example islacIQ1 (Glascock & Weickert 1998, Gene 223(1-2):221-231). Improvedrepression of the promoter during the growth phase results in improvedplasmid retention and higher cell density and, ultimately, in improvedprotein yield. For example, bacterial strains comprising the lacI^(q)gene overexpress the lacI repressor molecule and therefore preventformation of the recombinant protein during the growth phase moreefficiently than strains comprising the wildtype gene. In a preferredembodiment the gene causing overexpression of said lacI is lacIQ1 orlacI^(q), preferably lacI^(q). In a specifically preferred embodimentsaid bacterial host comprises the lacI^(q) gene on its chromosome.

In one embodiment said inducing of said promoter is performed with aninducer, wherein said inducer is preferably selected from IPTG andlactose, most preferably said inducer is lactose. Upon exposure of thebacterial host to an inducer, the repressor is inactivated and thepromoter becomes active. Addition of the strong inducer IPTG to theculture medium results in an immediate increase of the expression rateof the recombinant protein to a high level because IPTG directly entersthe cells by diffusion and binds and inactivates the active repressorInactivated lacI repressor molecules dissociates from the operator andallow high level transcription from the promoter. IPTG is notmetabolized by the cell and the transcription continues with high ratesuntil other metabolic parameters become limiting.

As mentioned before, high expression rates may lead to the formation ofinsoluble recombinant protein which is not capable of forming a VLP byself-assembly. Induction of protein expression with high concentrationsof IPTG is particularly prone to the formation of insoluble protein.Therefore, induction of the promoter is preferably achieved by theaddition of IPTG in concentrations which are below the concentrationwhich causes the expression to occur at its maximum rate (Kopetzki etal. 1989, Mol Gen Genet. 216:149-155).

In a preferred embodiment said inducing of said promoter is performedwith IPTG, wherein the concentration of said IPTG in said medium isabout 0.001 to 5 mM, preferably 0.001 to 1 mM, more preferably 0.005 to1 mM, still more preferably 0.005 to 0.5 mM. In a specifically preferredembodiment the concentration of said IPTG is about 0.01 mM, mostpreferrably 0.01 mM.

Alternatively, induction of the promoter is achieved by the addition oflactose. Induction of recombinant protein expression with lactoserequires that the bacterial host is capable of taking up lactose fromthe medium, e.g. by Lac permease and that it comprises β-galactosidaseactivity. The Lac permease dependent uptake of lactose into the cellsfollows a slower kinetic than the uptake of IPTG by diffusion.Furthermore, lactose does not directly interact with the lac operon butis converted by β-galactosidase to allolactose(1-6-O-β-galactopyranosyl-D-glucose) which is the actual inducer of thepromoter. Induction of recombinant protein expression by the addition oflactose is advantageous because it avoids the immediate increase of theexpression rate to a maximum level upon addition of the inducer and,thus, it reduces the risk of the formation of insoluble protein.

Allolactose is metabolised by the bacterial host during the productionphase and contributes carbon and metabolic energy to the bacterialmetabolism. This may further contribute to improved protein yield ascompared to induction with IPTG. Furthermore, induction by lactoseallows to a certain extend the control of the expression rate of therecombinant protein during the production phase via the lactoseconcentration in the medium. Induction by lactose is further preferredin a pharmaceutical production process because IPTG is expensive and isbelieved to be toxic. Its removal needs to be demonstrated at the end ofa the production process.

In a preferred embodiment said inducing of said promoter is performed bythe addition of lactose to said batch culture, wherein preferably saidbacterial host is capable of taking up lactose from the medium andwherein further preferably said bacterial host comprises β-galactosidaseactivity. Such bacteria strains can, for example, be obtained fromstrain collections such as ATTC (http://www.atcc.org). In a preferredembodiment, said bacterial host is an E. coli strain, preferably an E.coli strain selected from the group consisting of RB791, DH20, Y1088,W3110 and MG1655. Most preferably, said bacterial host is E. coli RB791.In a still more preferred embodiment said promoter is the tac promoteror mutant or variant thereof and said bacterial host is an E. colistrain which further comprises a gene causing overexpression of arepressor of the tac promoter, wherein said gene preferably is lacI^(q).The pH of the culture medium of the bacterial host can be controlledduring the fermentation process and regulated by the addition of acidicor alkaline solutions using methods which are well known in the art. Inone embodiment, said cultivating of said bacterial host and said feedingof said batch culture is performed under conditions, wherein the pH ofthe medium is controlled. In a preferred embodiment said pH is between5.5 and 8.0, more preferably between 6.5 and 7.5, even more preferablybetween 6.7 and 7.0 and most preferably said pH of said medium is 6.8.Said pH of said medium may be kept constant during the process or it mayfollow a certain profile during the different phases of the processwithin the pH ranges specified above. In a preferred embodiment said pHis kept constant at a value of 6.7 to 7.0, preferably said pH it is keptconstant at 6.8.

The process of the invention comprises a growth phase, wherein saidgrowth phase comprises a batch and a feed phase, wherein said growthphase and simultaneously said batch phase are initiated by saidcultivating said bacterial host and wherein said feed phase is initiatedby said feeding of said batch culture with said major carbon source.

The oxidative capacity of bacteria cells is limited and highconcentrations of the substrate may cause the formation of reducedproducts like acetate, which may lead to undesired acidification of themedium and to reduced growth of the bacteria. Therefore, the bacterialhost is grown in a fed-batch culture on a minimum medium with a limitedquantity of substrate. In one embodiment said cultivating of saidbacterial host is performed in a medium comprising said major carbonsource, wherein said medium preferably is a minimal medium, preferably achemically defined minimal medium. Most preferably said medium is R27medium as described in Example 5.

At the end of the batch phase, when the substrate contained in themedium is almost exhausted, medium containing the major carbon source(feed medium) is fed to said batch culture at the same rate as thedesired growth rate of the bacterial host, i.e. the growth rate of thebacterial host is limited by the feed rate of the substrate. It isunderstood by the skilled person that the decisive parameter is theactual mass flow of the substrate, preferably the major carbon source,and other nutrients required to maintain growth. Since in practice aconstant composition of the feed medium can be assumed, the flow raterefers to the volume flow of the medium. The same consideration appliesto the co-feed medium (see below).

Therefore, in one embodiment said feeding of said batch culture withsaid major carbon source is performed with a flow rate, wherein saidflow rate is limiting the growth rate of said bacterial host.

During the feed phase the growth rate can be freely selected in a widerange nearly up to the maximum growth rate (μ_(max)) if no inhibitionoccurs. The actual value of μ_(max) is highly dependent on the bacterialstrain, the expression construct and the growth conditions. The skilledperson will understand that the determination of μ_(max) is performedunder conditions under which the promoter is repressed.

For a given experimental set up, μ can be determined from the growthcurve of the culture by plotting biomass concentration (x) as determinedby OD₆₀₀ or cell wet weight (CWW) against the cultivation time anddetermining the exponential growth coefficient μ based on the equationx=x₀ e^(μt). The actual value of μ_(max) is determined as the growthrate μ of an exponentially growing batch culture in the beginning of thebatch phase when no substrate limitation occurs, i.e. without supply ofadditional medium by feeding. The growth rate μ can be determined bycomputing the ratio of the difference between natural logarithm of thetotal biomass X₂ measured at time t₂ and natural logarithm of the totalbiomass X₁ measured at time t₁ to the time difference (t₂−t₁):μ=(lnX₂=lnX₁)/(t₂−t₁).

Fed-batch culture allows the maintenance of a constant growth rate (μ).In a preferred embodiment the substrate, preferably the major carbonsource, is fed during the feed phase according to the exponentialincrease of the biomass (x). If during the feed phase the substrate issupplied at the same rate it is consumed, the culture is in a quasisteady state, analogous to the cultivation in a continuous culture.Because biomass formation and substrate consumption are correlated overthe substrate-referred yield coefficient Y_(x/s) (biomass [g]/substrate[g]), the substrate quantity (s) per time unit (t) to be supplied iscalculated according to the formula ds/dt=μ/Y_(x/s)x_(0 tot) e^(μt),wherein x_(0 tot) is the total biomass at feed start.

Therefore, in a preferred embodiment said feeding of said batch culturewith said major carbon source is performed with a flow rate, whereinsaid flow rate increases with an exponential coefficient μ, and whereinpreferably said exponential coefficient μ is below μ_(max). Thus, thegrowth rate of said bacterial host during the feed phase is set to avalue which is below μ_(max). In a preferred embodiment said exponentialcoefficient μ is about 30% to 70%, most preferably about 50% of μ_(max).In a specific embodiment of the invention μ is set to an absolute valueof 0.15 to 0.45 h⁻¹, more preferably 0.25 to 0.35 h⁻¹, most preferably μis 0.3 h⁻¹, provided that the set up of the process is such, that thesevalues are below μ_(max).

Bacteria are able to utilise a wide range of different substrates. Forthe purpose of the invention, preferred major carbon sources are glucoseand glycerol, preferably glycerol. Although the maximum specific growthrate (μ_(max)) of the expression host which can be achieved may behigher with glucose than with glycerol, glycerol causes less acetateformation and provides higher biomass yield per substrate (Y_(x/s)) and,ultimately, higher yield of the recombinant protein. Furthermore, thehandling of the liquid substrate glycerol is easier than that of solidcarbon sources like glucose which need to be dissolved in a separateprocess step.

As mentioned before, plasmid retention, i.e. the maintenance of theexpression plasmid in the bacterial host during the fermentationprocess, is essential for optimal yield of the recombinant protein.Plasmid retention can be assessed by spreading bacteria cells on a solidmedium to form single colonies and testing individual colonies for theirantibiotic resistance. For example, a plasmid retention of 100% meansthat 100 out of 100 tested colonies comprise the specific antibioticresistance which conferred by the expression plasmid. For the purpose ofthe invention plasmid retention at the end of the fermentation processis more than 80%, preferably more than 90%, more preferably more than95%, even more preferably more than 97% and most preferably 100%.

The optimal growth temperature of a bacterial strain is the temperatureat which it reaches its highest maximal growth rate (μ_(max)). Underotherwise not limiting conditions for most E. coli strains thistemperature is about 37° C. However, growth of the bacterial straincomprising the expression construct at the optimal growth temperatureand in the absence of a selective antibiotic may favour the loss of theexpression plasmid, whereas plasmid retention is generally improved whenthe expression strain is grown at lower temperature. Although themaximum growth rate of the expression strain is lower when the strain isgrown at temperatures below its optimal growth temperature as comparedto growth at the optimal growth temperature, the yield of recombinantprotein may be equal or even better at the lower temperature due toimproved plasmid retention.

In one embodiment of the invention, said cultivating of said bacterialhost and/or said feeding of said batch culture with said major carbonsource and/or said inducing said promoter with an inducer is thereforeperformed at a temperature below the optimal growth temperature of saidbacterial host. In a preferred embodiment said temperature is between 20and 37° C., preferably between 23 and 35° C., more preferably between 25and 33° C., even more preferably between 27 and 32° C., still morepreferably between 28 and 31° C. Still more preferably said temperatureis about 30° C., most preferably said temperature is 30° C.

The process of the invention comprises a production phase, wherein saidproduction phase is initiated by said inducing said promoter with aninducer. The time point for the initiation of said production phase canbe determined based on cultivation time and/or growth parameters.

The growth of the bacterial host during the fermentation process can beassessed by determining the optical density at 600 nm (OD₆₀₀), the cellwet weight (CWW [g/l]) and the cell dry weight (CDW [g/l]). Theseparameters can be used to define the optimal time point for the start ofthe production phase by addition of the inducer, preferably lactose, tothe medium. It is apparent for the skilled person, that on one handhigher CWW at the beginning of the production phase can be achieved byan extended feed phase and may lead to improved yield of the recombinantprotein but that on the other hand over-aged cultures may showinsufficient protein expression. The optimal time point for thebeginning of the production phase, which is initiated by said inducingof said promoter with an inducer, therefore needs to be determined forthe specific production conditions. For example, for expression of Qβ CPin E. coli RB791 in a total volume of 2 l, induction is started afterca. 14 h, when OD₆₀₀ has reached about 40 to 60. Surprisingly, similarparameters were found for the same process in a 50 l scale, whereinduction start is also after ca. 14 h when OD₆₀₀ has reached about 50.

Therefore, in one embodiment of the invention, said inducing of saidpromoter with said inducer is performed 10 h to 16 h after the beginningof said growth phase, preferably after 12 h to 15 h, more preferablyafter 13 h to 15 h, most preferably after about 14 h, wherein preferablysaid inducing of said promoter with said inducer is performed when theOD₆₀₀ has reached about 40 to 60, preferably about 50.

In a further embodiment, said inducing of said promoter with saidinducer is performed after an extended feed phase, wherein preferablysaid inducing of said promoter with said inducer is performed 14 h to 20h after the beginning of said cultivating of said bacterial host in amedium, preferably after 15 h to 18 h, more preferably after 16 h to 17h, most preferably after about 16.5 h, wherein preferably said inducingof said promoter with said inducer is performed when the OD₆₀₀ hasreached about 80 to 90, preferably about 85.

In one embodiment of the invention said inducing of said promoter withsaid inducer is performed when the OD₆₀₀ reached a value of 25 to 60,preferably 25 to 55, more preferably 30 to 50, most preferably 30 to 40.In a specifically preferred embodiment said inducing of said promoterwith said inducer is performed when OD₆₀₀ is 35.

In another embodiment of the invention said inducing of said promoterwith said inducer is performed after an extended feed phase, when theOD₆₀₀ reached a value of 60 to 120, preferably 70 to 110, morepreferably 80 to 100, most preferably 80 to 90. In a specificallypreferred embodiment the induction is started after and an extended feedphase when OD₆₀₀ is about 85, preferably 85.

Induction with IPTG: In one embodiment of the invention said inducing ofsaid promoter with an inducer is achieved by the addition of IPTG,wherein preferably said feeding of the culture with the major carbonsource is continued. Since IPTG is not metabolized by the bacterialhost, induction can be achieve by a single addition of IPTG to thedesired concentration. Alternatively, induction can be achieved by acontinuous flow of IPTG to the culture. In a preferred embodimentinduction is performed by addition of IPTG in a single addition or acontinuous flow, wherein said feeding of the batch culture with themajor carbon source is continued with a constant or an increasing flowrate of said major carbon source exponentially increasing flow rate ofthe major carbon source.

Induction with lactose: As described above, the induction of proteinexpression can alternatively be achieved by the addition of lactose tothe culture medium. In one embodiment of the invention, at the beginningof the production phase the exponential feed of the substrate isinterrupted and the culture is supplied with a constant flow ofinduction medium containing 100 to 300 g/l, preferably 100 g/l lactoseas the sole carbon source (lactose feed medium). Preferably, theconstant flow rate of lactose equals approximately the flow rate of thesubstrate at the end of the feed phase.

In a preferred embodiment of the invention said inducing of saidpromoter with an inducer is achieved by the addition of lactose, whereinpreferably said lactose is fed to said batch culture in a continuousflow during and wherein preferably said feeding of said batch culturewith said major carbon source is not continued.

Upon addition of lactose to the culture, the β-galactosidase activityincreases, lactose is converted to allolactose which induces the tacpromoter and the expression of the recombinant capsid is initiated. Inparallel, allolactose is further metabolised and contributes to theenergy supply for the bacterial host. The equilibrium of the feedingrate of the induction medium and the lactose consumption by the cellsthus determines the expression rate. The enzymatic reactions involved inthis cascade allow to control the process in such a way that theformation of inclusion bodies is minimised. The progress of inductionprocess can be monitored by determining the β-galactosidase activity inthe culture, e.g. by a β-Gal Assay Kit (Invitrogen, K1455-01).

In a more preferred embodiment of the invention said inducing of saidpromoter with an inducer is achieved by the addition of lactose, whereinpreferably said lactose is fed to said batch culture in a continuousflow during and wherein preferably said feeding of said batch culturewith said major carbon source is continued.

Discontinuous addition of inducer: Said inducer can be added to theculture discontinuously by a single addition at the beginning of theproduction phase or by a few subsequent additions during the productionphase. Discontinuous addition of the inducer, especially by a singleaddition is particularly suited when the inducer is IPTG since IPTG isnot metabolized by the bacterial host. Therefore, typically andpreferably no replacement of metabolised IPTG is necessary during theproduction phase. In one embodiment said inducing of said promoter withan inducer is performed by the addition of said inducer, preferably IPTGor lactose, most preferably IPTG, to said medium, wherein said induceris added to about its final concentration at once by a single additionat the beginning of the production phase, wherein preferably saidfeeding of said batch culture with said major carbon source iscontinued. In a preferred embodiment said inducing of said promoter withan inducer is performed by the addition of IPTG to said medium, whereinsaid IPTG is added to about its final concentration at once by a singleaddition, wherein preferably said feeding of said batch culture withsaid major carbon source is continued. Alternatively, said inducing ofsaid promoter with an inducer is performed by the addition of saidinducer, preferably IPTG or lactose, most preferably lactose, to saidmedium, wherein said addition is performed in several steps, preferablyin 1 to 5, more preferably in 2 to 4, most preferably in 3 steps duringthe production phase, wherein preferably said feeding of said batchculture with said major carbon source is continued.

Continuous addition (feeding) of inducer: Preferably, said inducer isadded to the medium in a continuous flow, preferably throughout theproduction phase. The continuous addition of the inducer is particularlysuited for lactose, since lactose is metabolised by the bacterial hostand therefore a continuous addition of lactose during the productionphase allows to maintain a lactose concentration in the medium whichallows for efficient induction of the promoter. In a preferredembodiment, said inducing of said promoter with an inducer is performedby feeding said batch culture with said inducer, wherein preferably saidinducer is IPTG or lactose, most preferably lactose, and wherein saidfeeding is performed in a continuous flow, wherein further preferablysaid feeding is performed throughout the production phase.

Co-feeding of inducer and major carbon source: The expression of therecombinant protein is an energy demanding process. To prevent yieldloss which might be caused by the excessive consumption of the inducerby the bacterial host and low expression rates resulting thereof, theculture can be additionally supplemented with substrate, preferably themajor carbon source, during the production phase, wherein the flow rateof inducer and/or the major carbon source is constant or increasing,preferably constant. When during the production phase the culture issupplemented with substrate at an increasing flow rate, the flow rate ispreferably increasing with an exponential rate.

Co-feeding with constant flow rate: In a preferred embodiment saidinducing of said promoter with an inducer is performed by co-feedingsaid batch culture with said inducer and said major carbon source,wherein said inducer is preferably IPTG or lactose, most preferablylactose, and wherein said major carbon source is glucose or glycerol,preferably glycerol, wherein said inducer, preferably lactose and saidmajor carbon source, preferably glycerol are co-fed to said batchculture at a flow rate, wherein said flow rate is preferably aboutconstant. In a further preferred embodiment said flow rate is chosen toallow feeding of said major carbon source to said batch culture at aboutthe same rate as at the end of the growth phase. In a still furtherpreferred embodiment said inducer, preferably lactose, and said majorcarbon source, preferably glycerol, are contained in the same medium(co-feed medium). In a further preferred embodiment said co-feed mediumis fed to said batch culture with a flow rate, wherein said flow rate ispreferably about constant, and wherein further preferably said flow rateis chosen to allow feeding of said major carbon source to said batchculture at about the same rate as at the end of the growth phase. In avery preferred embodiment said inducer is lactose and said major carbonsource is glycerol, wherein said lactose and said glycerol are co-fed tosaid batch culture in a ratio of about 2:1 to 1:4 (w/w).

In a further preferred embodiment of the invention lactose and saidmajor carbon source, preferably glycerol, are co-fed to said batchculture in a ration of 0:1 to 1:0 (w/w), preferably about 2:1 to about1:4 (w/w), more preferably about 1:1 to 1:3 (w/w), most preferably theratio is about 1:3 (w/w). In a preferred embodiment the ratio of lactoseand the major carbon source, preferably glycerol, is 1:1 (w/w). Inanother preferred embodiment the ratio of lactose and the major carbonsource, preferably glycerol, is 1:3 (w/w). In a more preferredembodiment said co-feed medium comprises ca. 200 g/l lactose and ca. 200g/l glycerol. In a still more preferred embodiment the co-feed mediumcomprises ca. 100 g/l lactose and ca. 300 g/l glycerol.

Co-feeding with increasing flow rate: Alternatively, said inducing ofsaid promoter with an inducer is performed by co-feeding said batchculture with said inducer and said major carbon source, wherein saidinducer is preferably IPTG or lactose, most preferably lactose, andwherein said major carbon source is glucose or glycerol, preferablyglycerol, wherein said inducer, preferably lactose and said major carbonsource, preferably glycerol are co-fed to said batch culture at a flowrate, wherein said flow rate is increasing, wherein said flow rate mayincrease with a linear or with an exponential characteristic, whereinpreferably the initial flow rate is chosen to to allow feeding of saidmajor carbon source to said batch culture at about the same rate as atthe end of the growth phase.

Further alternatively said inducing of said promoter with an inducer isperformed by co-feeding said batch culture with said inducer and saidmajor carbon source, wherein said inducer is preferably IPTG or lactose,most preferably lactose, and wherein said major carbon source is glucoseor glycerol, preferably glycerol, wherein said inducer, preferablylactose is fed to said batch culture at a first flow rate, and whereinsaid major carbon source, preferably glycerol is fed to said batchculture at a second flow rate, wherein said first flow rate is constantor increasing, preferably constant, and wherein said second flow rate isconstant or increasing, preferably increasing, wherein preferably theinitial value of said second flow rate is chosen to to allow feeding ofsaid major carbon source to said batch culture at about the same rate asat the end of the growth phase. In a very preferred embodiment saidinducer is lactose and said major carbon source is glycerol, whereinsaid lactose and said glycerol are co-fed to said batch culture in aratio of about 2:1 to 1:4 (w/w).

The growth of the bacterial host as determined by CDW, CWW or OD₆₀₀continues during the production phase at a growth rate which is lowerthan that during the growth phase and which is decreasing with theprocess time. In a further embodiment of the invention, said inducingsaid promoter with an inducer is performed by co-feeding said inducer,preferably lactose and said major carbon source, preferably glycerol, tosaid batch culture with an increasing flow rate, preferably with a flowrate wherein the incremental increase of the flow rate is adapted to theactual growth rate of the culture. In a further preferred embodimentsaid inducer, preferably lactose, and said major carbon source,preferably glycerol, are contained in the same medium (co-feed medium),wherein preferably the ratio between lactose and glycerol in said medium(co-feed medium) ranges from about 0:1 to 1:0 (w/w), preferably about2:1 to about 1:4 (w/w), more preferably about 1:1 to 1:3 (w/w), mostpreferably the ratio is about 1:3 (w/w). In a preferred embodiment theratio of lactose and the major carbon source, preferably glycerol, is1:1 (w/w). In another preferred embodiment the ratio of lactose and themajor carbon source, preferably glycerol, is 1:3 (w/w). In a morepreferred embodiment said medium (co-feed medium) comprises ca. 200 g/llactose and ca. 200 g/l glycerol. In a still more preferred embodimentthe induction medium comprises ca. 100 g/l lactose and ca. 300 g/lglycerol.

In one embodiment of the invention said inducing of said promoter withan inducer is performed by co-feeding said inducer, preferably lactoseand said major carbon source, preferably glycerol to said batch culture,wherein said inducer, preferably lactose and said major carbon source,preferably glycerol are contained in separate media which are separatelyfed to said culture.

At the end of the production phase the cells are harvested bycentrifugation. Typically, cells are harvested about 5 h after inductionstart, when a final OD₆₀₀ of 90 to 130 is reached. Further extension ofthe production phase leads to higher OD₆₀₀ and CWW values and thereforeto further improved yield of the expression construct.

Harvested cells may be suspended in a storage buffer and stored at −80°C. for further processing.

The total protein content of the cells is determined after cell lysis bySDS PAGE or LDS PAGE and comparison with a protein standard. The contentof soluble protein is determined by HPLC. The identity of the expressedcapsid protein is determined by western blotting. The concentration ofassembled VLPs can be analysed by size exclusion chromatography (Example18). VLP can preparatively be purified from lysed cells bychromatographic methods.

Scale-up of the process of the invention to large volumes is possiblewith only minor adaptations. The invention encompasses culture volumesin the range of 100 ml up to 6000 l. Preferred culture volumes are 40 to100 l, most preferably about 50 l. It is apparent for the skilled personthat larger culture volumes in particular require larger volumes of thepreculture which is used for inoculation. For example, a preculture maybe performed in two ore more steps with increasing preculture volume. Toensure plasmid retention in large culture volumes, the precultures whichare used as inoculum may contain an antibiotic to maintain selectionpressure. The skilled person is aware that plasmid retention can furtherbe improved by reducing the number of generations which is necessary toreach the desired final cell density. Therefore, it is advantageous toinoculate the precultures and the batch cultures with high celldensities. In a preferred embodiment the initial OD₆₀₀ of the precultureis 0.1 to 0.4, preferably about 0.3.

In one embodiment, prior to said cultivation step, said process furthercomprises the step of introducing said bacterial host into a medium,wherein said introducing is performed with an inoculum, wherein saidinoculum is produced in a preculture process comprising the step ofgrowing said bacterial host in a medium comprising an antibiotic,preferably kanamycin. More preferably, said preculture process comprisesthe steps of growing said bacterial host in a first medium comprising anantibiotic, preferably kanamycin, and diluting said first mediumcomprising the bacterial host with a second medium to an OD₆₀₀ of 0.1 to0.4, preferably about 0.3, wherein said second medium is essentiallyfree of an antibiotic, and further cultivating said bacterial host.

Furthermore, it is apparent for the skilled person, that thefermentation process of the invention is an aerobic process whichrequires adequate oxygen supply of the bacteria in the culture. Theoxygen demand of the bacterial host is, inter alia, increasing withincreasing cell density and increasing growth rate. Depending on thetotal volume and the oxygen demand of the bacterial host, oxygen can,for example, be supplied by stirring and/or by aeration with air.Alternatively, oxygen can also be supplied by aeration with pure oxygenor a mixture of pure oxygen with any other gas, preferably air, whereinpure oxygen refers to the technically pure gas as commonly available fortechnical purposes. A further possibility of supplying oxygen to thebacterial host is increasing the oxygen partial pressure in the mediumby increasing the pressure in the fermenter.

In a preferred embodiment of the invention, said cultivating saidbacterial host and/or said feeding of said batch culture and/or saidinducing of said promoter with an inducer is performed under conditions,wherein said bacterial host is supplied with oxygen, preferably byaeration with air, most preferably by aeration with air in a constantflow, wherein preferably said oxygen is supplied throughout the entireprocess, most preferably throughout the lag-, growth- and productionphase, and wherein further preferably the partial pressure of oxygen ismonitored in the culture medium and wherein the bacterial host isalternatively or additionally supplied with oxygen by aeration with pureoxygen, preferably when the partial pressure of oxygen in the medium(pO₂) is below a certain threshold. In a specifically preferredembodiment said threshold of pO₂ is in the range of 0% to 60%,preferably 10% to 50%, more preferably 20% to 45% most preferably saidthreshold is about 40%.

Oxygen supply, preferably by aeration with air and/or pure oxygen tomaintain the preferred pO₂ as described above, is routinely applied inthe process of the invention, preferably for culture volumes of 2 l andmore. Aeration with oxygen in the described manner is especiallypreferred in the scaled-up process, most preferably at 40 to 100 l andabove.

Therefore, one embodiment of the invention is a process for expressionof a recombinant capsid protein of a bacteriophage or a mutant orfragment thereof being capable of forming a VLP by self-assembly, saidprocess comprising the steps of: a) introducing an expression plasmidinto a bacterial host, wherein said expression plasmid comprises anexpression construct, wherein said expression construct comprises (i) afirst nucleotide sequence encoding said recombinant capsid protein, ormutant or fragment thereof, and (ii) a promoter being inducible bylactose; b.) cultivating said bacterial host in a medium comprising amajor carbon source; wherein said cultivating is performed in batchculture and under conditions under which said promoter is repressed bylacI, wherein said lacI is overexpressed by said bacterial host; c.)feeding said batch culture with said major carbon source; and d.)inducing said promoter with an inducer, wherein said feeding of saidbatch culture with said major carbon source is continued; whereinthroughout steps b.) to d.) of said process oxygen is supplied to saidbacterial host by a pO₂ in said medium of at least about 10% to 50%,preferably about 40%, and wherein further preferably said oxygen issupplied by aeration with air, pure oxygen, or a mixture of both,preferably by a mixture of air and pure oxygen.

EXAMPLES Example 1 Cloning Strategy for the Expression PlasmidpTac-nSd-Qb-mut (SEQ ID NO:1)

The coat protein-encoding gene (C) of E. coli RNA bacteriophage Qβ isamplified from plasmid pSDQb-mut (SEQ ID NO:33). The plasmid containsthe sequence of gene C coding for the 133-aa Qβ coat protein (CP) andthe 329-aa read through protein (A1). To prevent read-through,nucleotides 445-450 according to NCBI GenBank Acc. No. M99030 TGAACA(SEQ ID NO:31) are replaced by the sequence TAATGA (SEQ ED NO:32).

The coat protein-encoding gene C from plasmid pSDQb-mut is amplified byPCR. Oligonucleotide Qb-FOR3/2 (SEQ ID NO:34) with an internal EcoRIsite and a synthetic Shine-Dalgarno (SD, SEQ ID No:4) sequence annealsto the 5′ end of the Qβ CP gene. Oligonucleotide Qblang-REV2/2 (SEQ IDNO:35) contains an internal HindIII site and primes to the 3′ end of thenoncoding region of gene C. The 1054 by amplified PCR fragment includesnucleotides 46-1062 of NCBI GenBank Acc. No. M99039 (except thenucleotide changes described above) and the synthetic SD sequence. ThePCR fragment is digested with the restriction enzymes HindIII/EcoRI andthe resulting 1036 by fragment is inserted into the HindIII/EcoRIrestriction sites of a modified pKK223-3 vector (Pharmacia, NCBI GenBankAcc. No.: M77749, SEQ ID NO:27). In this modified pKK223-3 vector theampicillin resistance gene is replaced with the kanamycin resistancegene of vector pUC4K (Pharmacia, NCBI GenBank Acc. No.: X06404, SEQ IDNO:37).

Vector pTac-nSDQb-mut (SEQ ID NO:33) differs from vector pTacQb-mut inthe Shine-Dalgarno sequence. This Shine-Dalgarno sequence (nSD, SEQ IDNO:3) is introduced by amplifying the Qβ coat protein-encoding gene Cvia PCR from plasmid pTacQb-mut. Oligonucleotide nSDQb-mutEcoRIfor (SEQID NO:36) with an internal EcoRI site and the corresponding syntheticShine-Dalgarno (nSD) sequence anneals to the 5′ end of the Qβ CP gene.Oligonucleotide Qblang-REV2/2 (SEQ ID NO:35) contains an internalHindIII site and primes to the 3′ end of the noncoding region of gene C.The 1054 by amplified PCR fragment includes nucleotides 46-1062 of NCBIGenBank Acc. No. M99039 (except the nucleotide changes described above)and the synthetic nSD sequence. The PCR fragment is digested with therestriction enzymes HindIII/EcoRI and the resulting 1036 by fragment isinserted into the HindIII/EcoRI restriction sites of a modified pKK223-3vector (Pharmacia, NCBI GenBank Acc. No.: M77749, SEQ ID NO:27). In thismodified pKK223-3 vector the ampicillin resistance gene is replaced withthe kanamycin resistance gene of vector pUC4K (Pharmacia, NCBI GenBankAcc. No.: X06404, SEQ ID NO:37).

Example 2 Cloning Strategy for the Expression Plasmid pTac-nSD-AP205(SEQ ID NO:30)

The coat protein-encoding gene of Acinetobacter bacteriophage AP205 isamplified from plasmid pAP205-58. This plasmid contains the sequence ofthe coat protein gene (corresponding to nucleotides 1908-2303 of NCBIGenBank Acc. No. AF334111) coding for the 131-amino acid capsid proteinof bacteriophage AP205.

The coat protein-encoding gene is amplified by PCR. OligonucleotidenSDAP238-EcoRIfor (SEQ ID NO:38) with an internal EcoRI site and asynthetic Shine-Dalgarno (nSD) sequence anneals to the 5′ end of thecoat protein gene. Oligonucleotide AP238HindIIIrev (SEQ ID NO:39)contains an internal HindIII site and primes to the 3′ end of the coatprotein gene. This oligonucleotide introduces a second stop codon behindthe naturally occurring stop codon of the coat protein. The 438 byamplified PCR fragment includes nucleotides 1908-2303 of NCBI GenBankAcc. No. AF334111 and the synthetic nSD sequence. The PCR fragment isdigested with the restriction enzymes HindIII/EcoRI and the resulting420 by fragment is inserted into the HindIII/EcoRI restriction sites ofa modified pKK223-3 vector (Pharmacia, NCBI GenBank Acc. No.: M77749,SEQ ID NO:27). In this modified pKK223-3 vector the ampicillinresistance gene is replaced with the kanamycin resistance gene of vectorpUC4K (Pharmacia, NCBI GenBank Acc. No.: X06404, SEQ ID NO:37).

Example 3 Expression of Qβ CP Under Control of the Tac Promoter and nSD

The E. coli strain RB791 was transformed with plasmids pTac-nSD-Qb-mut(SEQ ID NO:1). The clone was grown in shake flasks. Each flask contained100 ml of R40 medium (main culture medium, Hypep 7455, glycerol, seeExample 5) with kanamycin (25 μg/ml) and was inoculated with over nightcultures at a start OD₆₀₀ of 0.3. The shake flasks were incubated for 4h (OD₆₀₀ between 4 and 5) at 30° C. and an agitation of 220 rpm. Theinduction was carried out with 0.5% of lactose for 4 h. Proteinproduction was determined by SDS-PAGE. The gel showed a strong proteinband which was identified as Qβ CP.

Example 4 Expression of AP205 CP Under Control of the Tac Promoter andSD Vs. nSD

9 clones of pTac-nSDAP205 (SEQ ID NO:30) and 6 clones of pTac-SDAP205were screened in shake flasks. pTac-SDAP205 (SEQ ID NO:40) is identicalto pTac-nSDAP205 but comprises the Shine-Dalgarno sequence of SEQ IDNO:4 instead of that of SEQ ID NO:3. Each flask contained 50 ml of R40medium (main culture medium, Hypep 7455, glycerol, see Example 5) withkanamycin (25 μg/ml) and was inoculated with over night cultures at astart OD₆₀₀ of 0.3 (for pTac-nSDAP205) or 0.4 (pTac-SDAP205). The shakeflasks were incubated for 4 h at 30° C. and an agitation of 220 rpm. Theinduction was carried out with 0.5% of lactose. Protein production wasdetermined by SDS-PAGE. For all tested clones expression of AP205 CP wassignificantly stronger from pTac-nSDAP205 than from pTac-SDAP205.

Example 5 Composition of Culture Media

Culture media were composed as described in Table 1.

TABLE 1 Composition of Culture media. Concentrations in [g/L] Main FeedInduction Main Main Medium + Medium + Medium + 20% Medium + Medium +Hypep + 50% Glycerol + 20% Bacto YE + Hypep Glycerol Glycerol LactoseGlycerol Component R27 R40 R41 R42 R43 Na₂HPO₄2H₂O 2.5 2.5 2.5 2.5 2.5KH₂PO₄ 3 3 3 3 3 K₂HPO₄ 5.2 5.2 5.2 5.2 5.2 Citrate 3.86 3.86 3.86 3.863.86 (NH₄)2SO₄ 4 4 4 4 4 Vit B1 0.01 0.01 0.02 0.02 0.01 CaCl₂2H₂O0.0147 0.0147 0.0147 0.0147 0.0147 MgSO₄7H₂O 0.5 0.5 9 9 0.5 FeCl₃6H₂O0.054 0.054 0.054 0.054 0.054 CoCl₂6H₂O 0.0005 0.0005 0.0005 0.00050.0005 MnCl₂4H₂O 0.003 0.003 0.003 0.003 0.003 CuCl₂2H₂O 0.0003 0.00030.0003 0.0003 0.0003 H₃BO₃ 0.003 0.003 0.003 0.003 0.003 Na₂MoO₄2H₂O0.0005 0.0005 0.0005 0.0005 0.0005 Zn(CH₃COO)₂2H₂O 0.0026 0.0026 0.00260.0026 0.0026 Glucose 5 — — — — Glycerol — 5 500 200 5 Lactose anhydrous— — — 200 — HyPep 7455 5 5 — — — Bacto Yeast Extract — — — — 5

Example 6 Expression of Qβ CP in a Fed-Batch Process (2 L Scale)

The fermentation process was performed in a bioreactor (Applikon 5 Ldished bottom) equipped with 2 disc stirrer (Ø 6 cm), baffles (3×16 cm),pH-, pO2-, and temperature control, and fermenter software BioXpertVersion 2.22

5 μL cryo culture of RB791 transformed with plasmids pTac-nSD-Qb-mutwere inoculated in 100 mL Erlenmeyer flasks containing 50 mL medium R40(25 μg/mL kanamycin) and cultivated for 14 h at 30° C. and 220 RPM overnight. After 14 h an OD₆₀₀ value of 6.0 was reached. For batchfermentation, 2 L of medium (R40) were pumped into the bioreactor. InTable 2 the cultivation parameters are listed.

TABLE 2 Parameter set points for batch phase. Parameter Set point UnitStirrer speed 1000 [rpm] Air supply 2.5 [L/min] O2-supply, maximal 2[L/min] Temperature 30 [° C.] O2-saturation >40 [%] pH 6.8 [—]

The bioreactor was inoculated with 100 mL inoculum. Samples of 2 mL weretaken, OD₆₀₀ determined and centrifuged at 14,000 RPM. Pellet andsupernatant were separated and frozen for further analysis. The biomassconcentration [g/L] was calculated using the following equation:

OD ₆₀₀×0.45[g×L⁻¹ ×OD ₆₀₀ ⁻¹]=biomass [g/L].

The Qbeta content in percent of the total protein content was calculatedas follows, assuming, that 50% of the E. coli biomass is protein:

Biomass [g×L⁻¹]/2=total protein [g×L⁻¹]

Qbeta [g×L⁻¹]/total protein [g×L⁻¹]×100=Qbeta/total protein [%].

In the fed-batch mode, which followed the batch mode, a feeding phasewas added. In the feeding phase substrate is supplied to the cells inthe reactor according to a defined profile. The feed profile depends onthe selected growth rate μ, the yield coefficient biomass to glycerol(Y_(x/glycerol)), the volume (Vf), and the concentration of substrate inthe feed (cf). substrate concentration. The feed was calculated usingthe following equation:

Feed Equation

mf=(μ/Y _(x/s) +m)Vf×Xf×e ^(μt)

pump=(mf/cf+b)/a

mf=mass flow [g/h]

μ=specific growth rate [l/h]

Y_(x/Glycerol)=Yield biomass to glycerol [g/g]

m=maintenance energy [g·g⁻¹·h⁻¹]

Vf=Volume at feed start

Xf=Biomass at feed start

cf=Concentration of substrate in feed [g/mL]

a+b=offset/slope of pump calibration equation

For the determination of the calibration parameters a and b, a pumpcalibration was carried out. In addition, the feed tube with feed bottlewas clamped into the feed pump and the pump was run with 7, 14 and 21%pump performance. The pumped feed volume per time was noted. In aresulting diagram of the relation of pump performance [%] to pumped feedsolution [mL/h], the slope (a) and the Y-axis section (b) wasdetermined. On the bioreactor the parameters in Table 3 were set forfed-batch cultivation.

TABLE 3 Parameters for fed-batch cultivation in bioreactor. ParameterSet point Unit Stirrer speed 1000 [rpm] Air supply 2.5 [L/min]O₂-supply, maximal 2 [L/min] Temperature 30 [° C.] O₂-saturation >40 [%]pH 6.8 [—]

After reaching a process time of approximately 7 h (end of batch) thefeed pump was turned on automatically. After further 7 h cultivation,when the OD₆₀₀ reached 55-60, the feed medium (for biomass propagation)was exchanged with the induction medium R42 (for biomass propagation andinduction). After 5 h feeding of R42 was stopped and the culture washarvested by centrifugation.

Analysis of Process Parameters:

The following process parameters were routinely analysed. The pO₂, pH,temperature and stirrer speed were measured online throughout theprocess time. The optical density was measured offline at 600 nm. Thedetermination of the β-galactosidase activity was performed using aβ-Gal Assay Kit (Invitrogen, cat. no. K1455-01). The activity wasspecified as units per mL OD₆₀₀=1.0. It is defined as the quantity ofOrtho-Nitrophenyl-β-D-Galactopyranosid (ONPG) in nmol, which ishydrolysed per minute and mL bacteria suspension (OD₆₀₀=1.0). Theaccumulated product was analysed by SDS-PAGE, the total protein content(soluble and insoluble protein) was determined and using HPLC analysis,the soluble fraction was measured. Cell disruption of E. coli wasperformed in lysis buffer (50 mM glucose, 25 mM tris/HCl (pH 8), 15 mMEDTA (pH 8.0)) with and ultrasonic homogeniser (Bandlin Sonoplus,HD2070). 250 μL bacteria suspension with an OD₆₀₀ of 50 were centrifugedwith 14000 RPM for 10 min. The pellet was resuspended in 250 μL, lysisbuffer (vortex) and placed at room temperature for 5 min. Afterwards,the cells were disrupted for 20 s with ultrasonic at 10% deviceperformance (cells on ice) and then the cell suspension was centrifugedat 14000 RPM, 10 min. The supernatant (soluble protein) was thenanalysed by SDS-PAGE and HPLC.

Samples before induction and at end of production (after 5 h induction)were taken from the bioreactor for analysis of Qβ formation analyzed bySDS-PAGE standardized to OD 5.0. At the end of cultivation, 1.9 l of theculture was harvested. After centrifugation, the following cell pelletswere obtained in three independent reactor runs: 1.) End OD₆₀₀ of 84:194 g CWW; 2.) End OD₆₀₀ of 88: 200 g CWW; 3.) End OD₆₀₀ of 86: 201 gCWW.

The plasmid retention in run 1 and 2 was 100% at induction start and100% at harvest. Based on comparison with a Qβ CP standard on SDS-PAGEthe yield was roughly estimated to be about 5 g/l Qβ CP. HPLC analysisrevealed a concentration of about 6 g/l Qβ VLP.

Example 7 Selection of Carbon Source and Bacterial Strain

Glucose and glycerol as carbon sources were compared. In order to testthe growth behaviour of each of the strains DH20 and RB791 on thesecarbon sources, shake flask experiments were conducted with mediumcontaining glucose (R27) and medium containing glycerol (R40). Bothmedia were supplemented with 25 μg/ml kanamycin. Each culture wasstarted with an initial OD₆₀₀ of 0.3. Induction was performed by adding0.5% lactose. The maximum specific growth rates (μ_(max)) and the yieldcoefficients (Y_(x/s)) were determined and are listed in Table 4. RB791grew faster on both, glucose and glycerol. In addition, the resultingyield coefficients were higher. Although glucose allowed higher maximumspecific growth rates (μ_(max)) the yield coefficients (Y_(x/s)) washigher for glycerol.

TABLE 4 Maximum specific growth rates and the yield coefficients of thecultivation experiments with RB791 and DH20 on glucose and glycerol.Value after 4.5 h Yield coefficient Culture Time Max. spec. (Y_(x/s))Carbon Acetate growth rate biomass from Strain source OD₆₀₀ [g l⁻¹](μ_(max)) [h⁻¹] substrate [g/g] RB791 glucose 6.24 0.44 0.71 0.72glycerol 4.04 0.21 0.62 0.86 DH20 glucose 2.52 0.42 0.51 0.71 glycerol2.82 0.25 0.50 0.81

Example 8 Determination of Optimal Temperature

The influence of temperature on product formation was investigated. Twoshake flask cultures were inoculated and incubated at 30° C. and 220rpm. After an OD₆₀₀ of 5 was reached, the cultures were induced withlactose. Subsequently, one culture was continued to be incubated at 37°C. and the other culture at 23° C. Results of the SDS-PAGE revealed thatexpression levels at 4 and 5 h after induction are higher in the cultureinduced at 37° C. Induction of the cultures for 19 h showed a higher Qβlevel in the cultures induced at 23° C.

Example 9 Induction by Co-Feed of Lactose and Glycerol

A feed solution of 20% glycerol and 20% lactose was composed (R42) andapplied to fermentation as described in Example 6 at induction start.FIG. 1 provides an overview over relevant process parameters throughoutthe entire process time. Expression was induced at 13.5 b at an OD₆₀₀ ofabout 55. Upon induction, the feed pump rate was set to constant.Glycerol did not accumulate with feeding. Lactose accumulated to 4 g/land then it started to diminish. The β-galactosidase activity rose to 10U/ml and decreased thereafter. Compared with the previous fermentationruns a.) lactose applied as a single lactose pulse at induction start,no feeding; b.) continuous lactose feed without glycerol, the activitywas with 7 U/ml higher and the maximum activity was already reachedafter 2 h as compared to 4 h in runs a.) and b.).

Example 10 Plasmid Retention

The effect of the following operating conditions on the plasmidretention was tested in the process described in Example 6: 1.)Preculture starting volume, 2) Kanamycin in the preculture, 3.) Growthand/or induction at 37° C. vs. 30° C. The results are summarised inTable 5. Precultures were started with volumes of 5 μl out of the cellbank vial. Inoculation of a small volume allowed growth of a precultureover-night. The preculture for QT0103_F8 contained 25 mg/l kanamycin,whereas the preculture for QT0103_F7 did not contain any kanamycin. Bothfermentations were operated at 30° C. and induced for 5 h. Judging fromthe plasmid retentions before and after 5 h induction, supplementing thepreculture with kanamycin has a positive effect on plasmid retention.Plasmid retention remained at 98% before and after 5 h induction. Incontrast, plasmid retentions reached only values of 80% when kanamycinwas omitted from the preculture. For a subsequent run, QT0203_F7, thepreculture was also started with 5 μl and grown in kanamycin containingmedium. The resulting fermentation in the bioreactor was operated at 37°C. from the beginning. Operation at 37° C. had a detrimental effect onthe plasmid stability. While the plasmid retention was at 99% beforeinduction, it dropped to 0% after 5 h induction. In order to testwhether a shorter preculture and thus, less generations, would improvethe plasmid retention after 5 h induction, a set of precultures werestarted with 300 μl volume from a thawed cell bank vial and grown inkanamycin free medium. Two fermenters were operated at 30° C. for thewhole run. An additional two fermenters were operated first at 30° C.for cell growth and than switched to 37° C. for the production phase.The resulting plasmid stabilities were all at 100% before and 5 h afterinduction.

TABLE 5 Summary of plasmid retention before and 5 h after inductionobtained under different operating conditions in terms of generations inthe preculture, with and without kanamycin in the preculture, and growthand/or induction at 37° C. Preculture Plasmid Plasmid Starting Kanamycinretention retention Culture in before after 5 h Bioreactor Volume pre-induc. induc. run [μl] culture [%] [%] Remarks QT0103_F8 5 25 mg/L 98 98whole process at 30° C. QT0103_F7 5 no 80 80 whole process at 30° C.QT0203_F7 5 25 mg/L 99 0 Bioreactor run at 37° C. QT0603_F7 300 no 100100 whole process at 30° C. QT0703_F8 300 no 100 100 Induction at 37°C., rest of the process at 30° C. QT0803_F9 300 no 100 100 whole processat 30° C. QT0903_F10 300 no 100 100 Induction at 37° C., rest of theprocess at 30° C.

Example 11 Variation in Time Point of Induction

In a process essentially as described in Example 6 the exponential feedprofile was programmed to start 7 h after the inoculation of thebioreactor. Under standard conditions, the scheduled time for inductionwas at 14 h process time. In order to test the effect of variations inthe time point of induction on the final cell densities, one culture wasinduced at 13.5 h (resulting in 6.5 h of exponential feed) and anotherculture at 14.5 h (resulting in 7.5 h of exponential feed). One cultureinduced at the regular 14 h time point served as a control (7 h ofexponential feed). Results are summarised in Table 6. Cell densityincreased with increasing length of feeding. Judged from a linearregression analysis of the available data points for final CWW, a linearrelationship appears to exist (r²=0.92).

TABLE 6 Variations in time point of induction: effect on final celldensity in terms of OD₆₀₀ and CWW. Process Time Duration of Point ofExp. Feed Final CWW Reactor Induction Phase [h] Final OD₆₀₀ [g/L] F2 13h 32 min 6.5 83.4 116.5 F1 14 h 02 min 7.0 82.4 122.5 F3 14 h 29 min 7.5100.4 141.1

Example 12 Variation in Time Point of Harvest

Harvest of the culture in a process essentially as described in Example6 is performed manually. Under standard conditions, the scheduled timefor harvest was at 19 h process time. The operation “Harvest” involvesthe manual ending of the bioreactor operations. In order to test theeffect of variations in the time point of harvest on the final celldensities, one culture was harvested at 18.8 h (resulting in 4.8 h ofinduction) and another culture at 19.5 h (resulting in 5.5 h ofinduction). One culture harvested at the regular 19 h time point servedas a control (5 h of induction). Results are summarized in Table 7. Celldensity increased with increasing length of induction because the cellsare still growing while induced.

TABLE 7 Variation in time point of harvest: effect on final cell densityin terms of OD600 and CWW. Process Time Length of Final CWW ReactorPoint of Harvest Induction [h] Final OD₆₀₀ [g/L] F5 18 h 50 min 4.8 91.4122.4 F4 19 h 00 min 5.0 92.2 127.5 F6 19 h 30 min 5.5 96.0 132.4

Example 13 Effect of Temperature

The effect of fermentation temperature in a process essentially asdescribed in Example 6 was investigated by running 6 fermentations at 5different temperature setpoints. Results are summarized in Table 8.Final cell densities were sensitive to the fermentation temperature withan optimum at a temperature of 30° C.

TABLE 8 Summarized results of different temperature setpoints on finalcell density in terms of OD600 and CWW. Temperature Final CWW Reactor [°C.] Final OD₆₀₀ [g/L] F5 25.0 37.8 62 F4 27.5 80.0 117 F3 30.0 92.8 123F4 30.0 92.4 125 F5 32.5 85.0 111 F6 35.0 79.6 107

Example 14 Scaled-Up Fermentation (50 l)

The process described in Example 6 was scaled up to a volume of 50 lorder to evaluate scale-up capability from the 2 L working volumebioreactor system to a larger volume. Key process parameters for thescaled-up process are summarized in Table 9.

TABLE 9 Process parameters of in 50 L bioreactor. Time Culture StepDescription [h] OD₆₀₀ Preculture 1 300 μl from cell bank vial aretransferred −11* 5.0 into 100 ml preculture medium contained in 500 mLshake flask and cultured for 16 h Preculture 2 Calculate the requiredvolume for transfer  −5* 4.0 in order to start with initial OD₆₀₀ of 0.3in 750 ml. Tranfer calculated volume (e.g. 50 ml) into 750 ml preculturemedium contained in 5000 mL shake flask Inoculation Pooled calculatedvolume (e.g. 1.4 L) is  0 of Bioreactor transferred into the 50 LBioreactor. Initial volume: = 40 L Induction The exponential feedingprofile is 14 46 Start switched to constant and feed is switched toinduction feed End of Culture is completed after 5 h of induction 19 128Culture *Relative to the time of bioreactor inoculation.

It was necessary to have two preculture expansion steps. In the firststep, the cells were expanded as established for the 2 L process(Example 6). After this step, cells were split into two 5000 ml shakeflask cultures, containing 750 ml medium each. Further expansion wasperformed for 5 h. The cultures in the 50 L bioreactors were performedwith the same time profile as in the 2 L system (Example 6). OD₆₀₀ atinduction start was 46, the final OD₆₀₀ was 128. Plasmid retention was100% before induction and 98% at the end of culture. The concentrationof Qβ CP protein in the medium at the end of culture was roughlyestimated 8 g/l using SDS-PAGE. The total amount of Qβ was estimatedabout 300 g for this reactor run.

Example 15 Effect of Extended Exponential Feed

The exponential feeding phase for fermentations performed according toExamples 6 or 14 was 7 h. After this time the cells reached a densityfor induction, which increased during induction to the targeted maximumOD₆₀₀ of around 100 to 130 as final cell density. Final OD₆₀₀, finalCWW, final CDW, plasmid retention at induction start and harvest and Qβconcentration at the end of culture are determined for reactor runsperformed as described in Examples 6 and 14, as in Example 14, whereinthe exponential feeding phase is extended to a duration up to 11 h,preferably to 10 h.

Example 16 Effect of Increased Feeding During Production

Example 9 demonstrates that the glycerol does not accumulate duringproduction phase, indicating that production might be limited by thefeeding rate of induction medium. Effect of extended feeding rate ofinduction medium on final OD₆₀₀, final CWW, final CDW, plasmid retentionat induction start and harvest and Qβ concentration at the end ofculture is determined in reactor runs as described in Example 6 and 14,preferably as in Example 14, wherein the feeding rate during productionis increased. Alternatively or additionally, the ratio between lactoseand glycerol in the feed medium shifted towards a higher glycerol and alower lactose concentration.

Example 17 HPLC Analysis of Qβ CP

Qβ CP was measured with an HPLC system as follows: A sample containingQβ CP was diluted appropriately in 1× reaction buffer (50 mMtris(hydroxymethyl)aminomethane buffer pH 8.0) containing 10 mM1,4-Dithio-DL-threitol and incubated for 15 min at 50° C. in athermomixer. After incubation the sample was centrifuged and thesupernatant was stored at 2° C. to 10° C. until HPLC analysis. 10 to 100μl of the sample were injected.

Qβ was quantified with a regression curve of known Qβ standardsregressed to the HPLC peak area detected at 215 nm after elution from aC₄ reversed phase column, 300 Å, 5 μm, 4.6×150 mm, Vydac Inc., Hesperia,USA (Cat No. 214TP5415) thermally equilibrated at 50° C. The flow ratethrough the system was 1 ml/min consisting of mobile phase A (0.12%trifluoroacetic acid in water) and mobile phase B (0.12% trifluoroaceticacid in acetonitrile) with the following gradient of phase B: 0 to 2 minconstant at 40%, 2 to 8 min linear increase to 50%, 8 to 10 min constantat 50%, 10 to 10.1 min linear decrease to 40%, and 10.1 to 12 minconstant at 40%.

Example 18 Determination of Qβ VLP by Analytical Size ExclusionChromatography

Analysis of Qβ particles by analytical size exclusion chromatography wasperformed using a TskgelG5000 PW_(XL-column) (10 μm, 7.8×300 mm, TosoHBiosep; Cat.-No. 08023) equilibrated in phosphate buffered saline (20 mMNa₂HPO₄/NaH₂PO₄, 150 mM NaCl pH 7.2). Elution was performed by anisocratic gradient for 20 min at 0.8 ml/min in phosphate bufferedsaline. The Qbeta concentration was determined from a regression curveof known Qβ standards regressed to the HPLC peak area detected at 260nm.

Example 19 Effect of Extended Exponential Feed

The exponential feeding phase for fermentations performed according toExamples 6 or 14 was 7 h. After this time the cells reached a densityfor induction, which increased during induction to the targeted maximumOD₆₀₀ of around 100 to 130 as final cell density. Final OD₆₀₀, finalCWW, plasmid retention before induction and at harvest and Qβconcentration at the end of culture were determined for reactor runsperformed as described in Examples 6 and 14, preferably as in Example14, wherein the exponential feeding phase was extended to a duration upto 12 h. In addition, the concentration of glycerol and lactose in theinduction feed were changed to 300 g/L and 100 g/L respectively. Theresults are summarized in Table 10.

TABLE 10 OD₆₀₀ and CWW at the end of cultivations, Plasmid Retentionbefore induction and at the end of cultivations as well as the peakoxygen mass flow. The cultivations were conducted with differentduration of exponential feeding. Plasmid Retention Duration [%] PeakOxygen Exponential OD₆₀₀ CWW before end of Mass Flow Feeding [h] [—][g/L] induction cultivation [vvm] 7 86 122 100 99 0.2 8 112 184 99 980.4 9 136 217 100 98 0.8 10 164 228 99 98 1.5 11 200 262 100 97 >4.5 1290 186 99 100 >4.5

According to LDS-PAGE analysis, the specific Qbeta concentration of allcultivations except for the cultivation with 12 h exponential feedingwas the same. An optimum regarding absolute Qbeta yield and oxygenconsumption was found for 9.5 h exponential feeding. Therefore, theprocess is preferably run with 9.5 h exponential feeding phase.

Example 20 Scaled Up Fermentation (50 L)

The process described in Example 6 and with 9.5 h exponential feedingphase with 300 g/L glycerol and 100 g/L lactose as described in Example19 was scaled up to a volume of 50 L in order to evaluate scale-upcapability from the 2 L working volume bioreactor system to a largervolume. Key process parameters for the scaled up process are summarizedin Table 11.

TABLE 11 Process parameters on the 50 L scale Time Culture StepDescription [h] Preculture 200 μl from cell bank vial were transferred−18*   into 800 ml preculture medium contained in 3000 mL shake flaskand cultured for 18 h (2 flasks) Inoculation Pooled total volume(approx. 1.6 L) was 0  of Bioreactor transferred into the 50 LBioreactor. Initial volume: = 35 L Induction The exponential feedingprofile was 16.5 Start switched to constant and feed was switched toinduction feed End of Culture was completed after 5 h of 21.5 Cultureinduction *Relative to the time of bioreactor inoculation.

It was necessary to change the preculture procedure in order toinoculate the larger reactor with approximately the same cell density.The cultures in the 50 L bioreactors were performed with the timeprofile optimised for the 2 L scale as described in Example 19. Thefinal cell wet weight for six cultivations was 188 g/L±9. Plasmidretention was 97.3%±1.4 at the end of culture. The concentration of QβCP protein in the medium at the end of culture was determined by C₄reversed phase HPLC (Example 17) to 10.8 g/L±0.3. The total amount of QβCP was 540 g for one 50 L run. The crude extract of approximately twotimes concentrated biomass was analysed for Qβ CP and Qβ VLP (Example18). The concentration of Qβ CP was 19.1 g/L±0.4 (C₄ reversed phaseHPLC), the concentration of Qβ VLP was 18.8 g/L±1.1. Therefore, theVLP-yield of the fermentation process is estimated to approximately 9-11g/l fermentation broth at the time of harvest.

1. A process for expression of a recombinant capsid protein of abacteriophage or a mutant or fragment thereof being capable of forming aVLP by self-assembly, said process comprising the steps of: a.)introducing an expression plasmid into a bacterial host, wherein saidexpression plasmid comprises an expression construct, wherein saidexpression construct comprises (i) a first nucleotide sequence encodingsaid recombinant capsid protein, or mutant or fragment thereof, and (ii)a promoter being inducible by lactose; b.) cultivating said bacterialhost in a medium comprising a major carbon source; wherein saidcultivating is performed in batch culture and under conditions underwhich said promoter is repressed by lacI, wherein said lad isoverexpressed by said bacterial host; c.) feeding said batch culturewith said major carbon source; and d.) inducing said promoter with aninducer, wherein said feeding of said batch culture with said majorcarbon source is continued.
 2. The process of claim 1, wherein saidbacteriophage is a RNA bacteriophage.
 3. (canceled)
 4. The process ofclaim 2, wherein said RNA bacteriophage is Qβ.
 5. The process of claim4, wherein said recombinant capsid protein has the amino acid sequenceof SEQ ID NO:5.
 6. (canceled)
 7. (canceled)
 8. The process of claim 1,wherein said expression construct comprises or alternatively consists ofthe nucleotide sequence of SEQ ID NO:6.
 9. The process of claim 1,wherein said expression plasmid comprises or preferably consists of thenucleotide sequence of SEQ ID NO:1.
 10. The process of claim 4, whereinsaid first nucleotide sequence is encoding a mutant coat protein of abacteriophage.
 11. The process of claim 1, wherein said promoter isselected from the group consisting of the a.) tac promoter; b.) trcpromoter; c.) tic promoter; d.) lac promoter; e.) lacUV5 promoter; f.)P_(syn) promoter; g.) lpp^(a) promoter; h.) lpp-lac promoter; i.) T7-lacpromoter; j.) T3-lac promoter; k.) T5-lac promoter; and l.) a promoterhaving at least 50% sequence homology to SEQ ID NO:2.
 12. The process ofclaim 1, wherein said promoter comprises the nucleotide sequence of SEQID NO:2.
 13. The process of claim 1, wherein said major carbon source isglycerol.
 14. The process of claim 1, wherein said feeding of said batchculture is performed with a flow rate, wherein said flow rate increaseswith an exponential coefficient μ, and wherein preferably saidexponential coefficient μ is below μ_(max).
 15. The process of claim 1,wherein said inducing of said promoter is performed by co-feeding saidbatch culture with said inducer and said major carbon source at aconstant flow rate, wherein preferably said inducer is lactose, andwherein further preferably said lactose and said major carbon source areco-fed to said batch culture in a ratio of about 2:1 to 1:4 (w/w). 16.The process of claim 1, wherein said inducing of said promoter isperformed by co-feeding said batch culture with said inducer and saidmajor carbon source at an increasing flow rate, wherein preferably saidinducer is lactose, and wherein further preferably said lactose and saidmajor carbon source are co-fed to said batch culture in a ratio of about2:1 to 1:4 (w/w).
 17. (canceled)
 18. The process of claim 1, whereinsaid inducer is IPTG and wherein preferably the concentration of saidIPTG in said medium is 0.001 to 5 mM.
 19. (canceled)
 20. The process ofclaim 1, wherein said lacI is overexpressed by said bacterial host,wherein said overexpression is caused by lacI^(q) or lacQ1, preferablyby lacI^(q).
 21. (canceled)
 22. (canceled)
 23. The process of claim 1,wherein said inducer is lactose and wherein said bacterial hostcomprises β-galactosidase activity.
 24. The process of claim 1, whereinsaid cultivating and said feeding of said batch culture and saidinducing of said promoter is performed at a temperature which is belowthe optimal growth temperature of said bacterial host.
 25. The processof claim 9 wherein: a.) said major carbon source is glycerol; b.) saidfeeding of said batch culture is performed with a flow rate, whereinsaid flow rate increases with an exponential coefficient μ, and whereinpreferably said exponential coefficient μ is below μ_(max); c.) saidinducer is lactose; d.) and said lactose and said major carbon sourceare co-fed to said batch culture in a ratio of 2:1 to 1:4 (w/w),preferably 1:1 to 1:3 (w/w), most preferably 1:3 (w/w); e.) saidbacterial host is E. coli RB791; and f.) said cultivating and feeding ofsaid batch culture and said inducing of said promoter is performed at atemperature of about 30° C.
 26. The process of claim 1 wherein: a.) saidexpression plasmid comprises or preferably consists of the nucleotidesequence of SEQ ID NO:30; b.) said major carbon source is glycerol; c.)said feeding of said batch culture is performed with a flow rate,wherein said flow rate increases with an exponential coefficient μ, andwherein preferably said exponential coefficient μ is below μ_(max); d.)said inducer is lactose; e.) said lactose and said major carbon sourceare co-fed to the batch culture in a ratio of 2:1 to 1:4 (w/w),preferably 1:1 to 1:3 (w/w), most preferably 1:3 (w/w); f.) saidbacterial host is E. coli RB791; and g.) said cultivating and feeding ofsaid batch culture and said inducing of said promoter is performed at atemperature of about 30° C.
 27. The process of claim 1, whereinthroughout steps b.) to d.) of said process oxygen is supplied to saidbacterial host by a pO₂ in the medium of at least about 40%.